Pakistan Institute of Medical Sciences

The rhizosphere is undoubtedly the most complex microhabitat, comprised of an integrated network of plant roots, soil, and a diverse consortium of bacteria, fungi, eukaryotes, and archaea. The rhizosphere conditions have a direct impact on crop growth and yield. Nutrient-rich rhizosphere environments stimulate plant growth and yield and vice versa. Extensive cultivation exhaust most of the soils which need to be nurtured before or during the next crop. Chemical fertilizers are the major source of crop nutrients but their uncontrolled and widespread usage has posed a serious threat to the sustainability of agriculture and stability of an ecosystem. These chemicals are accumulated in the soil, drained in water, and emitted to the air where they persist for decades causing a serious threat to the overall ecosystem. Plant growth-promoting rhizobacteria (PGPR) present in the rhizosphere convert many plant-unavailable essential nutrients e.g., nitrogen, phosphorous, zinc, etc. into available forms. PGPR produces certain plant growth hormones (such as auxin, cytokinin, and gibberellin), cell lytic enzymes (chitinase, protease, hydrolases, etc.), secondary metabolites, and antibiotics, and stress alleviating compounds (e.g., 1-Aminocyclopropane-1- carboxylate deaminase), chelating agents (siderophores), and some signaling compounds (e.g., N-Acyl homoserine lactones) to interact with the beneficial or pathogenic counterparts in the rhizosphere. These multifarious activities of PGPR improve the soil structure, health, fertility, and functioning which directly or indirectly support plant growth under normal and stressed environments. Rhizosphere engineering with these PGPR has a wide-ranging application not only for crop fertilization but developing eco-friendly sustainable agriculture. Due to severe climate change effects on plants and rhizosphere biology, there is growing interest in stress-resilient PGPM and their subsequent application to induce stress (drought, salinity, and heat) tolerance mechanism in plants. This review describes the three components of rhizosphere engineering with an explicit focus on the broader perspective of PGPM that could facilitate rhizosphere engineering in selected hosts to serve as an efficient component for sustainable agriculture.

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs.

BACKGROUND: For more than three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has provided a framework to quantify health loss due to diseases, injuries, and associated risk factors. This paper presents GBD 2023 findings on disease and injury burden and risk-attributable health loss, offering a global audit of the state of world health to inform public health priorities. This work captures the evolving landscape of health metrics across age groups, sexes, and locations, while reflecting on the remaining post-COVID-19 challenges to achieving our collective global health ambitions. METHODS: The GBD 2023 combined analysis estimated years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs) for 375 diseases and injuries, and risk-attributable burden associated with 88 modifiable risk factors. Of the more than 310 000 total data sources used for all GBD 2023 (about 30% of which were new to this estimation round), more than 120 000 sources were used for estimation of disease and injury burden and 59 000 for risk factor estimation, and included vital registration systems, surveys, disease registries, and published scientific literature. Data were analysed using previously established modelling approaches, such as disease modelling meta-regression version 2.1 (DisMod-MR 2.1) and comparative risk assessment methods. Diseases and injuries were categorised into four levels on the basis of the established GBD cause hierarchy, as were risk factors using the GBD risk hierarchy. Estimates stratified by age, sex, location, and year from 1990 to 2023 were focused on disease-specific time trends over the 2010-23 period and presented as counts (to three significant figures) and age-standardised rates per 100 000 person-years (to one decimal place). For each measure, 95% uncertainty intervals [UIs] were calculated with the 2·5th and 97·5th percentile ordered values from a 250-draw distribution. FINDINGS: Total numbers of global DALYs grew 6·1% (95% UI 4·0-8·1), from 2·64 billion (2·46-2·86) in 2010 to 2·80 billion (2·57-3·08) in 2023, but age-standardised DALY rates, which account for population growth and ageing, decreased by 12·6% (11·0-14·1), revealing large long-term health improvements. Non-communicable diseases (NCDs) contributed 1·45 billion (1·31-1·61) global DALYs in 2010, increasing to 1·80 billion (1·63-2·03) in 2023, alongside a concurrent 4·1% (1·9-6·3) reduction in age-standardised rates. Based on DALY counts, the leading level 3 NCDs in 2023 were ischaemic heart disease (193 million [176-209] DALYs), stroke (157 million [141-172]), and diabetes (90·2 million [75·2-107]), with the largest increases in age-standardised rates since 2010 occurring for anxiety disorders (62·8% [34·0-107·5]), depressive disorders (26·3% [11·6-42·9]), and diabetes (14·9% [7·5-25·6]). Remarkable health gains were made for communicable, maternal, neonatal, and nutritional (CMNN) diseases, with DALYs falling from 874 million (837-917) in 2010 to 681 million (642-736) in 2023, and a 25·8% (22·6-28·7) reduction in age-standardised DALY rates. During the COVID-19 pandemic, DALYs due to CMNN diseases rose but returned to pre-pandemic levels by 2023. From 2010 to 2023, decreases in age-standardised rates for CMNN diseases were led by rate decreases of 49·1% (32·7-61·0) for diarrhoeal diseases, 42·9% (38·0-48·0) for HIV/AIDS, and 42·2% (23·6-56·6) for tuberculosis. Neonatal disorders and lower respiratory infections remained the leading level 3 CMNN causes globally in 2023, although both showed notable rate decreases from 2010, declining by 16·5% (10·6-22·0) and 24·8% (7·4-36·7), respectively. Injury-related age-standardised DALY rates decreased by 15·6% (10·7-19·8) over the same period. Differences in burden due to NCDs, CMNN diseases, and injuries persisted across age, sex, time, and location. Based on our risk analysis, nearly 50% (1·27 billion [1·18-1·38]) of the roughly 2·80 billion total global DALYs in 2023 were attributable to the 88 risk factors analysed in GBD. Globally, the five level 3 risk factors contributing the highest proportion of risk-attributable DALYs were high systolic blood pressure (SBP), particulate matter pollution, high fasting plasma glucose (FPG), smoking, and low birthweight and short gestation-with high SBP accounting for 8·4% (6·9-10·0) of total DALYs. Of the three overarching level 1 GBD risk factor categories-behavioural, metabolic, and environmental and occupational-risk-attributable DALYs rose between 2010 and 2023 only for metabolic risks, increasing by 30·7% (24·8-37·3); however, age-standardised DALY rates attributable to metabolic risks decreased by 6·7% (2·0-11·0) over the same period. For all but three of the 25 leading level 3 risk factors, age-standardised rates dropped between 2010 and 2023-eg, declining by 54·4% (38·7-65·3) for unsafe sanitation, 50·5% (33·3-63·1) for unsafe water source, and 45·2% (25·6-72·0) for no access to handwashing facility, and by 44·9% (37·3-53·5) for child growth failure. The three leading level 3 risk factors for which age-standardised attributable DALY rates rose were high BMI (10·5% [0·1 to 20·9]), drug use (8·4% [2·6 to 15·3]), and high FPG (6·2% [-2·7 to 15·6]; non-significant). INTERPRETATION: Our findings underscore the complex and dynamic nature of global health challenges. Since 2010, there have been large decreases in burden due to CMNN diseases and many environmental and behavioural risk factors, juxtaposed with sizeable increases in DALYs attributable to metabolic risk factors and NCDs in growing and ageing populations. This long-observed consequence of the global epidemiological transition was only temporarily interrupted by the COVID-19 pandemic. The substantially decreasing CMNN disease burden, despite the 2008 global financial crisis and pandemic-related disruptions, is one of the greatest collective public health successes known. However, these achievements are at risk of being reversed due to major cuts to development assistance for health globally, the effects of which will hit low-income countries with high burden the hardest. Without sustained investment in evidence-based interventions and policies, progress could stall or reverse, leading to widespread human costs and geopolitical instability. Moreover, the rising NCD burden necessitates intensified efforts to mitigate exposure to leading risk factors-eg, air pollution, smoking, and metabolic risks, such as high SBP, BMI, and FPG-including policies that promote food security, healthier diets, physical activity, and equitable and expanded access to potential treatments, such as GLP-1 receptor agonists. Decisive, coordinated action is needed to address long-standing yet growing health challenges, including depressive and anxiety disorders. Yet this can be only part of the solution. Our response to the NCD syndemic-the complex interaction of multiple health risks, social determinants, and systemic challenges-will define the future landscape of global health. To ensure human wellbeing, economic stability, and social equity, global action to sustain and advance health gains must prioritise reducing disparities by addressing socioeconomic and demographic determinants, ensuring equitable health-care access, tackling malnutrition, strengthening health systems, and improving vaccination coverage. We live in times of great opportunity. FUNDING: Gates Foundation and Bloomberg Philanthropies.

This paper describes the evolving role of robotics in healthcare and allied areas with special concerns relating to the management and control of the spread of the novel coronavirus disease 2019 (COVID-19). The prime utilization of such robots is to minimize person-to-person contact and to ensure cleaning, sterilization and support in hospitals and similar facilities such as quarantine. This will result in minimizing the life threat to medical staff and doctors taking an active role in the management of theCOVID-19 pandemic. The intention of the present research is to highlight the importance of medical robotics in general and then to connect its utilization with the perspective of COVID-19 management so that the hospital management can direct themselves to maximize the use of medical robots for various medical procedures. This is despite the popularity of telemedicine, which is also effective in similar situations. In essence, the recent achievement of the Korean and Chinese health sectors in obtaining active control of the COVID-19 pandemic was not possible without the use of state of the art medical technology.

BACKGROUND: Neonatal sepsis is a primary cause of neonatal mortality and is an urgent global health concern, especially within low-income and middle-income countries (LMICs), where 99% of global neonatal mortality occurs. The aims of this study were to determine the incidence and associations with neonatal sepsis and all-cause mortality in facility-born neonates in LMICs. METHODS: The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) study recruited mothers and their neonates into a prospective observational cohort study across 12 clinical sites from Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Data for sepsis-associated factors in the four domains of health care, maternal, birth and neonatal, and living environment were collected for all mothers and neonates enrolled. Primary outcomes were clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality in neonates during the first 60 days of life. Incidence proportion of livebirths for clinically suspected sepsis and laboratory-confirmed sepsis and incidence rate per 1000 neonate-days for all-cause mortality were calculated. Modified Poisson regression was used to investigate factors associated with neonatal sepsis and parametric survival models for factors associated with all-cause mortality. FINDINGS: Between Nov 12, 2015 and Feb 1, 2018, 29 483 mothers and 30 557 neonates were enrolled. The incidence of clinically suspected sepsis was 166·0 (95% CI 97·69-234·24) per 1000 livebirths, laboratory-confirmed sepsis was 46·9 (19·04-74·79) per 1000 livebirths, and all-cause mortality was 0·83 (0·37-2·00) per 1000 neonate-days. Maternal hypertension, previous maternal hospitalisation within 12 months, average or higher monthly household income, ward size (>11 beds), ward type (neonatal), living in a rural environment, preterm birth, perinatal asphyxia, and multiple births were associated with an increased risk of clinically suspected sepsis, laboratory-confirmed sepsis, and all-cause mortality. The majority (881 [72·5%] of 1215) of laboratory-confirmed sepsis cases occurred within the first 3 days of life. INTERPRETATION: Findings from this study highlight the substantial proportion of neonates who develop neonatal sepsis, and the high mortality rates among neonates with sepsis in LMICs. More efficient and effective identification of neonatal sepsis is needed to target interventions to reduce its incidence and subsequent mortality in LMICs. FUNDING: Bill & Melinda Gates Foundation.

The traditional healthcare system is at the doorstep for entering into the arena of molecular medicine. The enormous knowledge and ongoing research have now been able to demonstrate methodologies that can alter DNA coding. The techniques used to edit or change the genome evolved from the earlier attempts like nuclease technologies, homing endonucleases, and certain chemical methods. Molecular techniques like meganuclease, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) initially emerged as genome-editing technologies. These initial technologies suffer from lower specificity due to their off-targets side effects. Moreover, from biotechnology’s perspective, the main obstacle was to develop simple but effective delivery methods for host cell entry. Later, small RNAs, including microRNA (miRNA) and small interfering RNA (siRNA), have been widely adopted in the research laboratories to replace lab animals and cell lines. The latest discovery of CRISPR/Cas9 technology seems more encouraging by providing better efficiency, feasibility, and multi-role clinical application. This later biotechnology seem to take genome-engineering techniques to the next level of molecular engineering. This review generally discusses the various gene-editing technologies in terms of the mechanisms of action, advantages, and side effects. The traditional healthcare system is at the doorstep for entering into the arena of molecular medicine. The enormous knowledge and ongoing research have now been able to demonstrate methodologies that can alter DNA coding. The techniques used to edit or change the genome evolved from the earlier attempts like nuclease technologies, homing endonucleases, and certain chemical methods. Molecular techniques like meganuclease, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) initially emerged as genome-editing technologies. These initial technologies suffer from lower specificity due to their off-targets side effects. Moreover, from biotechnology’s perspective, the main obstacle was to develop simple but effective delivery methods for host cell entry. Later, small RNAs, including microRNA (miRNA) and small interfering RNA (siRNA), have been widely adopted in the research laboratories to replace lab animals and cell lines. The latest discovery of CRISPR/Cas9 technology seems more encouraging by providing better efficiency, feasibility, and multi-role clinical application. This later biotechnology seem to take genome-engineering techniques to the next level of molecular engineering. This review generally discusses the various gene-editing technologies in terms of the mechanisms of action, advantages, and side effects. Over the last half century after post-DNA helical structure discovery, the world has seen a continuous staircase outburst of various molecular technologies, which are now heading forward toward translation into clinical and laboratory practice.1Friedrich M. Ueber die chemische Zusammensetzung der Eiterzellen [On the chemical composition of pus cells].Medicinisch-chemische Untersuchungen. 1871; 4: 441-460Google Scholar Given the availability of sequencing platforms, acquired wisdom about the micro-mechanics at work within the genetic apparatus, and the introduction of user-friendly nanotechnologies, it was possible for next-generation scientists to manipulate the genetic codes at various levels.2Watson J.D. Crick F.H.C. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (8367) Google Scholar Over the last two decades we saw a plethora of molecular techniques, which allowed us to edit genes or their alter pathways, allowing humans for the first time to micro-edit the DNA codes and further to alter the mRNA fate through post-transcriptional modifications.3Sanger F. Coulson A.R. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase.J. Mol. Biol. 1975; 94: 441-448Crossref PubMed Scopus (1587) Google Scholar Principally, genome-wide editing techniques can be interpreted as methods where DNA sequences are changed by deletions, mRNA processing, and post-transcriptional modifications to result in altered gene expression, leading to functional behavior of proteins.4Mullis K.B. https://www.karymullis.com/Date: 2016Google Scholar, 5Pavletich N.P. Pabo C.O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A.Science. 1991; 252: 809-817Crossref PubMed Scopus (1744) Google Scholar Common to these methods are three basic steps, including mechanisms for genetic tool entry into the cell and later nucleus; altering gene transcription and onward processing function; and, finally, the end-output in the shape of a suppressed, overexpressed, or simply an altered protein product.6Claussin C. Chang M. Multiple Rad52-Mediated Homology-Directed Repair Mechanisms Are Required to Prevent Telomere Attrition-Induced Senescence in Saccharomyces cerevisiae.PLoS Genet. 2016; 12: e1006176Crossref PubMed Scopus (16) Google Scholar, 7Arai N. Kagawa W. [Molecular mechanisms of homologous recombination promoted by budding yeast Rad52].Seikagaku. 2014; 86: 693-697PubMed Google Scholar From a holistic point of view, the techniques involve an apparently simplistic concept involving multiple receptor-ligand interactions; varying cell entry modes like lipofection, sonification, and transfection; and further downstream pathway effects. Furthermore, these technologies are variable in terms of their specificity and sensitivity, off-target effects, finances, and technique expertise. The body’s immune response to accept the foreign genetic elements within the cells can lead to the rejection of foreign tissues. Moreover, molecular knowledge, in terms of methodology differences, defining targetable diseases, innovative nanotechnology tools for gene editing, and ethical aspects, also needs to be understood. The platforms for these technologies are improving every day, with a plethora of new data appearing due to technology miniaturization and automation and newer discoveries to improve the yield and specificity of an edited product. Alongside the developmental improvement in genome-wide engineering the regulatory work-up, standardization protocols need to be devised to reduce inter and intra-method imprecision, defining the indications and contraindications of every technique to help improve the concept of personalized medicine. This review briefly explains the available technologies, provides comparison and contrast between different genome-editing methods, and identifies some newer versions of genome editing with possible bioethical concerns. PubMed searches with the keywords genome-editing techniques or gene-editing techniques in the last 10 and 5 years yielded a total of 4,466 and 4,054 references, except some historical and related references. Specific searches for articles dealing with specific genome-editing methods included conventional genome-editing systems (n = 100), chemical methods (n = 252), meganucleases (n = 83), zinc-finger nucleases (ZFNs) (n = 890), transcription activator-like effector nucleases (TALENs) (n = 1,136), homing endonucleases (n = 265), and CRISPR (n = 11,421). The search was therefore limited to reviews showing conceptual information of common techniques and comparative information about ZFNs, TALENSs, and CRISPR technologies. Finally, the literature was searched for learning newer and advanced gene-editing methods and bioethical concerns associated with genome biotechnologies. The recent expansion and advancements in the field of biotechnology provided us with information and insight into the biochemical and molecular mechanisms to edit DNA and, thus, modify downstream pathways. To date, multiple biotechnologies have shown promise for clinical use, but the field of genome-editing technologies is rapidly evolving and improving. The new techniques seem promising, but the earlier ones have also been updated and improved. For simplicity and consolidation, an overview of genome-editing techniques is presented in Figure 1. Representative genome-editing techniques are discussed below.(1)Conventional genome-editing technique. In the true sense, the technique may not relate with evolving genome-editing techniques. As highlighted in Figure 1, it includes homologous recombination related with gene intervention. While not much in vogue or lab use today, the technique is based on physiological processes involving a double-stranded repair system. However, some recent data have shown RAD52 protein to be important in mediating homologous recombination, and this protein therefore has been considered as a therapy target in certain cancers like BRCA 1 and 2 repair pathways.6Claussin C. Chang M. Multiple Rad52-Mediated Homology-Directed Repair Mechanisms Are Required to Prevent Telomere Attrition-Induced Senescence in Saccharomyces cerevisiae.PLoS Genet. 2016; 12: e1006176Crossref PubMed Scopus (16) Google Scholar, 7Arai N. Kagawa W. [Molecular mechanisms of homologous recombination promoted by budding yeast Rad52].Seikagaku. 2014; 86: 693-697PubMed Google Scholar However, the technique as of now could not gain widespread introduction due to the emergence of newer techniques.(2)Chemical modalities of genome editing. Komiyama8Komiyama M. Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.Artif. DNA PNA XNA. 2014; 5: e1112457Crossref PubMed Scopus (6) Google Scholar utilized non-restriction enzyme methodology termed artificial restriction DNA cutter (ARCUT). This method uses pseudo-complementary peptide nucleic acid (pcPNA), whose job is to specify the cleavage site within the chromosome or the telomeric region. Once pcPNA specifies the site, excision here is carried out by cerium (CE) and EDTA (chemical mixture), which performs the splicing function.8Komiyama M. Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.Artif. DNA PNA XNA. 2014; 5: e1112457Crossref PubMed Scopus (6) Google Scholar Furthermore, the technology uses a DNA ligase that can later attach any desirable DNA within the spliced site. The advantage of this particular technique is that it can be used in high salt concentrations. Upon initial introduction, the technique looked quite appealing to the clinical market; however, later issues like increased turnaround time and specifically the manufacturing of site-specific pcPNA became huge hurdles (Figure 2).8Komiyama M. Chemical modifications of artificial restriction DNA cutter (ARCUT) to promote its in vivo and in vitro applications.Artif. DNA PNA XNA. 2014; 5: e1112457Crossref PubMed Scopus (6) Google Scholar, 9Ito K. Komiyama M. Site-selective scission of human genome using PNA-based artificial restriction DNA cutter.Methods Mol. Biol. 2014; 1050: 111-120Crossref PubMed Scopus (7) Google Scholar(3)Homing endonuclease systems. Homing endocucleases (HEs) with this word “homing” practically is interpreted as lateral transmission of a genome DNA sequence. The general concept involves a DNA segment where a site is removed by the endocnulceases, which thus results in the formation of 2 segments of DNA fragment.10Schultz B.R. Chamberlain J.S. Recombinant adeno-associated virus transduction and integration.Mol. Ther. 2008; 16: 1189-1199Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar So what are these HEs? They are nucleases that occur naturally, with a size almost equivalent to 14 bp, and they are capable of splicing slightly larger DNA sequences.11Smith J. Grizot S. Arnould S. Duclert A. Epinat J.C. Chames P. Prieto J. Redondo P. Blanco F.J. Bravo J. et al.A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.Nucleic Acids Res. 2006; 34: e149Crossref PubMed Scopus (246) Google Scholar Recently, the introduction of recombinant adeno-associated viruses (rAAVs) have allowed them as efficient vehicles for transporting genetic tools of genome engineering into the cell, as depicted in Figure 3.12Yoon Y. Wang D. Tai P.W.L. Riley J. Gao G. Rivera-Pérez J.A. Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.Nat. Commun. 2018; 9: 412Crossref PubMed Scopus (41) Google Scholar Issues pertaining to this technology include engineering difficulties in the preparation of these nucleases as well as developing vectors for their entry into cells.13Rey-Rico A. Cucchiarini M. Controlled release strategies for rAAV-mediated gene delivery.Acta Biomater. 2016; 29: 1-10Crossref PubMed Scopus (37) Google Scholar Another issue with rAAV, though improving with better biotechnology, was off-target effects like reducing site specificity, less DNA integration, and possible host genome mutations.14Jin L. Lange W. Kempmann A. Maybeck V. Günther A. Gruteser N. Baumann A. Offenhäusser A. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.J. Biotechnol. 2016; 233: 171-180Crossref PubMed Scopus (9) Google ScholarFigure 3Schematic Showing within with and with DNA for include the entry of into cell, by and within release of for entry into delivery of homing and desirable DNA of the DNA and (6) desirable DNA of the Figure nuclease systems. These systems nuclease for DNA editing. The common techniques are termed molecular DNA these are that are in the to of DNA sequences was as a genetic tool to modify This genetic has been in by the to create as for DNA These meganucleases are by to create like and which can further site-specific M. C. P. Duclert A. of meganucleases using a learning 2014; PubMed Scopus (9) Google Scholar The technique two basic first is the of a cleavage site, and endonucleases out the K. 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OBJECTIVES: We conducted a Pakistan-wide community-based survey on the prevalence of type 2 diabetes using glycated haemoglobin (HbA1c) as the screening test. The aim was to estimate diabetes prevalence across different demographic groups as well as all regions of Pakistan. DESIGN, SETTINGS AND PARTICIPANTS: Multistaged stratified cluster sampling was used for the representative selection of people aged ≥20 years, residing in 378 sampled clusters of 16 randomly selected districts, in this cross-sectional study. Eligible participants had blood drawn for HbA1c analyses at field clinics near to their homes. The oral glucose tolerance test (OGTT) was conducted on a subsample of the participants. Overall and stratified prevalence of type 2 diabetes and its association with risk factors were estimated using logistic regression models. MAIN OUTCOME MEASURES: Prevalence of prediabetes and type 2 diabetes. RESULTS: Of 18 856 eligible participants the prevalence of prediabetes was 10.91% (95% CI 10.46 to 11.36, n=2057) and type 2 diabetes was 16.98% (95% CI 16.44 to 17.51, n=3201). Overall, the mean HbA1c level was 5.62% (SD 1.96), and among newly diagnosed was 8.56% (SD 2.08). The prevalence was highest in age 51-60 years (26.03%, p<0.001), no formal education (17.66%, p<0.001), class III obese (35.09%, p<0.001), family history (31.29%, p<0.001) and female (17.80%, p=0.009). On multivariate analysis, there was a significant association between type 2 diabetes and older age, increase in body mass index and central obesity, positive family history, and having hypertension and an inverse relation with education as a categorical variable. On a subsample (n=1027), summary statistics for diagnosis of diabetes on HbA1c showed a sensitivity of 84.7%, specificity of 87.2% and area under the receiver operating characteristic curve 0.86, compared with OGTT. CONCLUSIONS: The prevalence of type 2 diabetes and prediabetes is much higher than previously thought in Pakistan. Comprehensive strategies need to be developed to incorporate screening, prevention and treatment of type 2 diabetes at a community level.

The prevalence of rheumatic diseases in developing countries is largely unknown. Studies which allow comparison of data within the contrasting communities of the Third World and the developed world have the potential to provide insights into disease aetiologies. The current study compared the frequency of rheumatic symptoms (point prevalence) amongst 1997 adults distributed evenly between poor rural and poor urban communities and relatively affluent urban people. Comparisons were also made with similarly but previously derived prevalence rates of rheumatic symptoms and rheumatoid arthritis (RA) in south Pakistan and Pakistanis in England. A significantly higher prevalence of joint pain was seen in the north compared with the south. RA was more common in the north and similar to the frequency amongst Pakistanis resident in England. Ethnic and genetic susceptibility might have accounted for this. There was significantly more soft-tissue rheumatism and back pain in the northern rural population compared with those in the city. Fibromyalgia was almost completely absent from the urban affluent, but osteoarthritis of the knee was significantly more common in this community, perhaps due to relative obesity. RA was least in the urban poor, a phenomenon that might be attributable to earlier death of females or other undetermined factors.

Improving infant and young child feeding practices will help South Asian countries achieve the Millennium Development Goal of reducing child mortality. This paper aims to compare key indicators of complementary feeding and their determinants in children aged 6-23 months across five South Asian countries - Bangladesh, India, Nepal, Pakistan and Sri Lanka. The latest Demographic and Health Survey and National Family Health Survey India data were used. The analyses were confined to last-born children aged 6-23 months - 1728 in Bangladesh, 15,028 in India, 1428 in Nepal, 2106 in Sri Lanka and 443 infants aged 6-8 months in Pakistan. Introduction of solid, semi-solid or soft foods, minimum dietary diversity, minimum meal frequency and minimum acceptable diet, and their significant determinants were compared across the countries. Minimum dietary diversity among children aged 6-23 months ranged from 15% in India to 71% in Sri Lanka, with Nepal (34%) and Bangladesh (42%) in between. Minimum acceptable diet among breastfed children was 9% in India, 32% in Nepal, 40% in Bangladesh and 68% in Sri Lanka. The most consistent determinants of inappropriate complementary feeding practices across all countries were the lack of maternal education and lower household wealth. Limited exposure to media, inadequate antenatal care and lack of post-natal contacts by health workers were among predictors of inappropriate feeding. Overall, complementary feeding practices among children aged 6-23 months need improvement in all South Asian countries. More intensive interventions are necessary targeting the groups with sup-optimal practices, while programmes that cover entire populations are being continued.

BACKGROUND: Twin pregnancy was associated with significantly higher rates of adverse neonatal and perinatal outcomes, especially for the second twin. In addition, the maternal complications (potentially life-threatening conditions-PLTC, maternal near miss-MNM, and maternal mortality-MM) are directly related to twin pregnancy and independently associated with adverse perinatal outcome. The objective of the preset study is to evaluate perinatal outcomes associated with twin pregnancies, stratified by severe maternal morbidity and order of birth. METHODS: Secondary analysis of the WHO Multicountry Survey on Maternal and Newborn Health (WHOMCS), a cross-sectional study implemented in 29 countries. Data from 8568 twin deliveries were compared with 308,127 singleton deliveries. The occurrence of adverse perinatal outcomes and maternal complications were assessed. Factors independently associated with adverse perinatal outcomes were reported with adjusted PR (Prevalence Ratio) and 95%CI. RESULTS: The occurrence of severe maternal morbidity and maternal death was significantly higher among twin compared to singleton pregnancies in all regions. Twin deliveries were associated with higher rates of preterm delivery (37.1%), Apgar scores less than 7 at 5th minute (7.8 and 10.1% respectively for first and second twins), low birth weight (53.2% for the first and 61.1% for the second twin), stillbirth (3.6% for the first and 5.7% for the second twin), early neonatal death (3.5% for the first and 5.2% for the second twin), admission to NICU (23.6% for the first and 29.3% for the second twin) and any adverse perinatal outcomes (67% for the first twin and 72.3% for the second). Outcomes were consistently worse for the second twin across all outcomes. Poisson multiple regression analysis identified several factors independently associated with an adverse perinatal outcome, including both maternal complications and twin pregnancy. CONCLUSION: Twin pregnancy is significantly associated with severe maternal morbidity and with worse perinatal outcomes, especially for the second twin.

Intellectual disability (ID) is a clinically and genetically heterogeneous disorder, affecting 1-3% of the general population. Although research into the genetic causes of ID has recently gained momentum, identification of pathogenic mutations that cause autosomal recessive ID (ARID) has lagged behind, predominantly due to non-availability of sizeable families. Here we present the results of exome sequencing in 121 large consanguineous Pakistani ID families. In 60 families, we identified homozygous or compound heterozygous DNA variants in a single gene, 30 affecting reported ID genes and 30 affecting novel candidate ID genes. Potential pathogenicity of these alleles was supported by co-segregation with the phenotype, low frequency in control populations and the application of stringent bioinformatics analyses. In another eight families segregation of multiple pathogenic variants was observed, affecting 19 genes that were either known or are novel candidates for ID. Transcriptome profiles of normal human brain tissues showed that the novel candidate ID genes formed a network significantly enriched for transcriptional co-expression (P<0.0001) in the frontal cortex during fetal development and in the temporal-parietal and sub-cortex during infancy through adulthood. In addition, proteins encoded by 12 novel ID genes directly interact with previously reported ID proteins in six known pathways essential for cognitive function (P<0.0001). These results suggest that disruptions of temporal parietal and sub-cortical neurogenesis during infancy are critical to the pathophysiology of ID. These findings further expand the existing repertoire of genes involved in ARID, and provide new insights into the molecular mechanisms and the transcriptome map of ID.

AIMS: To assess the prevalence and management of depressive disorders in people with Type 2 diabetes in different countries. METHODS: People with diabetes aged 18-65 years and treated in outpatient settings were recruited in 14 countries and underwent a psychiatric interview. Participants completed the Patient Health Questionnaire and the Problem Areas in Diabetes scale. Demographic and medical record data were collected. RESULTS: A total of 2783 people with Type 2 diabetes (45.3% men, mean duration of diabetes 8.8 years) participated. Overall, 10.6% were diagnosed with current major depressive disorder and 17.0% reported moderate to severe levels of depressive symptomatology (Patient Health Questionnaire scores >9). Multivariable analyses showed that, after controlling for country, current major depressive disorder was significantly associated with gender (women) (P<0.0001), a lower level of education (P<0.05), doing less exercise (P<0.01), higher levels of diabetes distress (P<0.0001) and a previous diagnosis of major depressive disorder (P<0.0001). The proportion of those with either current major depressive disorder or moderate to severe levels of depressive symptomatology who had a diagnosis or any treatment for their depression recorded in their medical records was extremely low and non-existent in many countries (0-29.6%). CONCLUSIONS: Our international study, the largest of this type ever undertaken, shows that people with diabetes frequently have depressive disorders and also significant levels of depressive symptoms. Our findings indicate that the identification and appropriate care for psychological and psychiatric problems is not the norm and suggest a lack of the comprehensive approach to diabetes management that is needed to improve clinical outcomes.

Anemia during pregnancy and the postpartum period is commonly caused by iron deficiency and is a significant worldwide issue with severe consequences for both mother and developing fetus. From a worldwide perspective, iron-deficiency anemia (IDA) during pregnancy is highest in the Asia-Pacific region; however, there has been little guidance in this region for safe and effective treatment. An expert panel was convened to develop a concise and informative set of recommendations for the treatment of IDA in pregnant and postpartum women in the Asia-Pacific region. This manuscript provides these recommendations and aims to reduce the morbidity and mortality associated with IDA in pregnant and postpartum women in the Asia-Pacific region. The consensus recommendations define anemia as a hemoglobin (Hb) level <10.5 g/dL during pregnancy and <10 g/dL during the postpartum period, and provide cut-off Hb levels to initiate therapy with oral iron, intravenous iron or red blood cell transfusion.

chief constituents against COVID-19. Among the studied compounds, we found that dithymoquinone (DTQ), with binding affinity of -8.6 kcal/mol compared to a positive control (chloroquine, -7.2 kcal/mol) , has the high potential of binding at SARS-CoV-2:ACE2 interface and thus could be predicted as a plausible inhibitor to disrupt viral-host interactions. Molecular dynamics simulation of 100 ns well complemented binding affinity of the compound and revealed strong stability of DTQ at the docked site. Additionally, MM-PBSA also affirms the docking results. Compound DTQ of the present study, if validated in wet lab experiments, could be used to treat COVID-19 and could serve as a lead in the future for development of more effective natural antivirals against COVID-19. Communicated by Ramaswamy H. Sarma.

OBJECTIVES: To evaluate the chest radiographs of children diagnosed with non-severe pneumonia on the basis of the current World Health Organization guidelines (fast breathing alone) for radiological evidence of pneumonia. DESIGN: Descriptive analysis. SETTING: Outpatient departments of six hospitals in four cities in Pakistan. PARTICIPANTS: 2000 children with non-severe pneumonia were enrolled; 1932 children were selected for chest radiography. INTERVENTIONS: Two consultant radiologists used standardised WHO definitions to evaluate chest radiographs; no clinical information was made available to them. If they disagreed, the radiographs were read by a third radiologist; the final classification was based on agreement between two of the three radiologists. MAIN OUTCOME MEASURES: Presence or absence of pneumonia on radiographs. RESULTS: Chest radiographs were reported normal in 1519 children (82%). Radiological evidence of pneumonia was reported in only 263 (14%) children, most of whom had interstitial pneumonitis. Lobar consolidation was present in only 26 children. The duration of illness did not correlate significantly with the presence of radiological changes (relative risk 1.17, 95% confidence interval 0.91 to 1.49). CONCLUSION: Most children diagnosed with non-severe pneumonia on the basis of the current WHO definition had normal chest radiographs.

BACKGROUND: Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin-gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. METHODS: In BARNARDS, consenting mother-neonates aged 0-60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic-pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. FINDINGS: Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin-gentamicin, ceftazidime-amikacin, piperacillin-tazobactam-amikacin, and amoxicillin clavulanate-amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime-amikacin than for neonates treated with ampicillin-gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14-0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin-gentamicin; 286 (73·3%) to amoxicillin clavulanate-amikacin; 301 (77·2%) to ceftazidime-amikacin; and 312 (80·0%) to piperacillin-tazobactam-amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin-gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate-amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime-amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin-tazobactam-amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis. INTERPRETATION: Our data raise questions about the empirical use of combined ampicillin-gentamicin for neonatal sepsis in LMICs because of its high resistance and high rates of frequency of resistance and low probability of target attainment. Accessibility and affordability need to be considered when advocating antibiotic treatments with variance in economic health structures across LMICs. FUNDING: The Bill & Melinda Gates Foundation.

BACKGROUND: Knowledge about coronary heart disease (CHD) and its risk factors is an important pre-requisite for an individual to implement behavioral changes leading towards CHD prevention. There is scant data on the status of knowledge about CHD in the general population of Pakistan. The objective of this study was to assess knowledge of CHD in a broad Pakistani population and identify the factors associated with knowledge. METHODS: Cross sectional study was carried out at four tertiary care hospitals in Pakistan using convenience sampling. Standard questionnaire was used to interview 792 patient attendants (persons accompanying patients). Knowledge was computed as a continuous variable based on correct answers to fifteen questions. Multivariable linear regression was conducted to determine the factors independently associated with knowledge. RESULTS: The mean age was 38.1 (+/- 13) years. 27.1% had received no formal education. The median knowledge score was 3.0 out of a possible maximum of 15. Only 14% were able to correctly describe CHD as a condition involving limitation in blood flow to the heart. Majority of respondents could identify only up to two risk factors for CHD. Most commonly identified risk factors were stress (43.4%), dietary fat (39.1%), smoking (31.9%) and lack of exercise (17.4%). About 20% were not able to identify even a single risk factor for CHD. Factors significantly associated with knowledge included age (p = 0.023), income (p < 0.001), education level (p < 0.001), residence (p < 0.001), a family history of CHD (p < 0.001) and a past history of diabetes (p = 0.004). Preventive practices were significantly lacking; 35%, 65.3% and 84.6% had never undergone assessment of blood pressure, glucose or cholesterol respectively. Only a minority felt that they would modify their diet, stop smoking or start exercising if a family member was to develop CHD. CONCLUSION: This is the first study assessing the state of CHD knowledge in a relatively diverse non-patient population in Pakistan. There are striking gaps in knowledge about CHD, its risk factors and symptoms. These translate to inadequate preventive behavior patterns. Educational programs are urgently required to improve the level of understanding of CHD in the Pakistani population.

NIMA related Kinases (NEK7) plays an important role in spindle assembly and mitotic division of the cell. Over expression of NEK7 leads to the progression of different cancers and associated malignancies. It is becoming the next wave of targets for the development of selective and potent anti-cancerous agents. The current study is the first comprehensive computational approach to identify potent inhibitors of NEK7 protein. For this purpose, previously identified anti-inflammatory compound i.e., Phenylcarbamoylpiperidine-1,2,4-triazole amide derivatives by our own group were selected for their anti-cancer potential via detailed Computational studies. Initially, the density functional theory (DFT) calculations were carried out using Gaussian 09 software which provided information about the compounds' stability and reactivity. Furthermore, Autodock suite and Molecular Operating Environment (MOE) software's were used to dock the ligand database into the active pocket of the NEK7 protein. Both software performances were compared in terms of sampling power and scoring power. During the analysis, Autodock results were found to be more reproducible, implying that this software outperforms the MOE. The majority of the compounds, including M7, and M12 showed excellent binding energies and formed stable protein-ligand complexes with docking scores of - 29.66 kJ/mol and - 31.38 kJ/mol, respectively. The results were validated by molecular dynamics simulation studies where the stability and conformational transformation of the best protein-ligand complex were justified on the basis of RMSD and RMSF trajectory analysis. The drug likeness properties and toxicity profile of all compounds were determined by ADMETlab 2.0. Furthermore, the anticancer potential of the potent compounds were confirmed by cell viability (MTT) assay. This study suggested that selected compounds can be further investigated at molecular level and evaluated for cancer treatment and associated malignancies.

BACKGROUND: Anxiety disorder, one of the highly disabling, prevalent and common mental disorders, is known to be more prevalent in persons with type 2 diabetes mellitus (T2DM) than the general population, and the comorbid presence of anxiety disorders is known to have an impact on the diabetes outcome and the quality of life. However, the information on the type of anxiety disorder and its prevalence in persons with T2DM is limited. AIMS: To assess the prevalence and correlates of anxiety disorder in people with type 2 diabetes in different countries. METHODS: People aged 18-65 years with diabetes and treated in outpatient settings were recruited in 15 countries and underwent a psychiatric interview with the Mini-International Neuropsychiatric Interview. Demographic and medical record data were collected. RESULTS: A total of 3170 people with type 2 diabetes (56.2% women; with mean (SD) duration of diabetes 10.01 (7.0) years) participated. The overall prevalence of anxiety disorders in type 2 diabetic persons was 18%; however, 2.8% of the study population had more than one type of anxiety disorder. The most prevalent anxiety disorders were generalised anxiety disorder (8.1%) and panic disorder (5.1%). Female gender, presence of diabetic complications, longer duration of diabetes and poorer glycaemic control (HbA1c levels) were significantly associated with comorbid anxiety disorder. A higher prevalence of anxiety disorders was observed in Ukraine, Saudi Arabia and Argentina with a lower prevalence in Bangladesh and India. CONCLUSIONS: Our international study shows that people with type 2 diabetes have a high prevalence of anxiety disorders, especially women, those with diabetic complications, those with a longer duration of diabetes and poorer glycaemic control. Early identification and appropriate timely care of psychiatric problems of people with type 2 diabetes is warranted.

// Faheem Ahmed Khan 1 , Nuruliarizki Shinta Pandupuspitasari 1 , Huang Chun-Jie 1 , Zhou Ao 1 , Muhammad Jamal 2 , Ali Zohaib 3 , Farhan Ahmed Khan 4 , Muthia Raihana Hakim 5 and Zhang ShuJun 1 1 Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Education Ministry of China, Huazhong Agricultural University, Wuhan, People&rsquo;s Republic of China 2 State Key Laboratory of Agriculture Microbiology, Huazhong Agricultural University, Wuhan, People&rsquo;s Republic of China 3 Key Laboratory of Special Pathogens and Center for Emerging Infectious Diseases, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China 4 Department of Cardiovascular Medicine, Zulfiqar Ali Bhutto Medical University, Pakistan Institute of Medical Sciences, Islamabad, Pakistan 5 Tongji Medical College, Huazhong University of Science And Technology, Wuhan, China Correspondence to: Zhang ShuJun, email: // Keywords : CRISPR/Cas9, genetic diseases, cancer, genome editing, next generation sequencing Received : March 12, 2016 Accepted : May 19, 2016 Published : May 26, 2016 Abstract Cancer is caused by a series of alterations in genome and epigenome mostly resulting in activation of oncogenes or inactivation of cancer suppressor genes. Genetic engineering has become pivotal in the treatment of cancer and other genetic diseases, especially the formerly-niche use of clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9. In defining its superior use, we have followed the recent advances that have been made in producing CRISPR/Cas9 as a therapy of choice. We also provide important genetic mutations where CRISPRs can be repurposed to create adaptive immunity to fight carcinomas and edit genetic mutations causing it. Meanwhile, challenges to CRISPR technology are also discussed with emphasis on ability of pathogens to evolve against CRISPRs. We follow the recent developments on the function of CRISPRs with different carriers which can efficiently deliver it to target cells; furthermore, analogous technologies are also discussed along CRISPRs, including zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). Moreover, progress in clinical applications of CRISPR therapeutics is reviewed; in effect, patients can have lower morbidity and/or mortality from the therapeutic method with least possible side-effects.