Lemuel Shattuck Hospital

BACKGROUND: SAMtools and BCFtools are widely used programs for processing and analysing high-throughput sequencing data. They include tools for file format conversion and manipulation, sorting, querying, statistics, variant calling, and effect analysis amongst other methods. FINDINGS: The first version appeared online 12 years ago and has been maintained and further developed ever since, with many new features and improvements added over the years. The SAMtools and BCFtools packages represent a unique collection of tools that have been used in numerous other software projects and countless genomic pipelines. CONCLUSION: Both SAMtools and BCFtools are freely available on GitHub under the permissive MIT licence, free for both non-commercial and commercial use. Both packages have been installed >1 million times via Bioconda. The source code and documentation are available from https://www.htslib.org.

Type 2 Diabetes Mellitus (T2DM), one of the most common metabolic disorders, is caused by a combination of two primary factors: defective insulin secretion by pancreatic β-cells and the inability of insulin-sensitive tissues to respond appropriately to insulin. Because insulin release and activity are essential processes for glucose homeostasis, the molecular mechanisms involved in the synthesis and release of insulin, as well as in its detection are tightly regulated. Defects in any of the mechanisms involved in these processes can lead to a metabolic imbalance responsible for the development of the disease. This review analyzes the key aspects of T2DM, as well as the molecular mechanisms and pathways implicated in insulin metabolism leading to T2DM and insulin resistance. For that purpose, we summarize the data gathered up until now, focusing especially on insulin synthesis, insulin release, insulin sensing and on the downstream effects on individual insulin-sensitive organs. The review also covers the pathological conditions perpetuating T2DM such as nutritional factors, physical activity, gut dysbiosis and metabolic memory. Additionally, because T2DM is associated with accelerated atherosclerosis development, we review here some of the molecular mechanisms that link T2DM and insulin resistance (IR) as well as cardiovascular risk as one of the most important complications in T2DM.

Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association. The Structural Variation Analysis Group of The 1000 Genomes Project reports an integrated structural variation map based on discovery and genotyping of eight major structural variation classes in 2,504 unrelated individuals from across 26 populations; structural variation is compared within and between populations and its functional impact is quantified. The Structural Variation Analysis Group of The 1000 Genomes Project reports an integrated structural variation map based on discovery and genotyping of eight major structural variation classes in genomes for 2,504 unrelated individuals from across 26 populations. They characterize structural variation within and between populations and quantify its functional effect. The authors further create a phased reference panel that will be valuable for population genetic and disease association studies.

Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

Abstract Ocular dominance columns were examined by a variety of techniques in juvenile macaque monkeys in which one eye had been removed or sutured closed soon after birth. In two monkeys the removal was done at 2 weeks and the cortex studied at 1 1/2 years. Physiological recordings showed continuous responses as an electrode advanced along layer IV C in a direction parallel to the surface. Examination of the cortex with the Fink-Heimer modification of the Nauta method after lesions confined to single lateral-geniculate layers showed a marked increase, in layer IV G, in the widths of columns belonging to the surviving eye, and a corresponding shrinkage of those belonging to the removed eye. Monocular lid closures were made in one monkey at 2 weeks of age, for a period of 18 months, in another at 3 weeks for 7 months, and in a third at 2 days for 7 weeks. Recordings from the lateral geniculate body showed brisk activity from the deprived layers and the usual abrupt eye transitions at the boundaries between layers. Cell shrinkage in the deprived layers was moderate - far less severe than that following eye removal, more marked ipsilaterally than contralaterally, and more marked the earlier the onset of the deprivation. In autoradiographs following eye injection with a mixture of tritiated proline and tritiated fucose the labelling of terminals was confined to geniculate layers corresponding to the injected eye. Animals in which the open eye was injected showed no hint of invasion of terminals into the deprived layers. Similarly in the tectum there was no indication of any change in the distribution of terminals from the two eyes. The autoradiographs of the lateral geniculates provide evidence for several previously undescribed zones of optic nerve terminals, in addition to the six classical subdivisions. In the cortex four independent methods, physiological recording, transneuronal autoradiography, Nauta degeneration, and a reduced-silver stain for normal fibres, all agreed in showing a marked shrinkage of deprived-eye columns and expansion of those of the normal eye, with preservation of the normal repeat distance (left-eye column plus right-eye column). There was a suggestion that changes in the columns were more severe when closure was done at 2 weeks as opposed to 3, and more severe on the side ipsilateral to the closure. The temporal crescent representation in layer IV C of the hemisphere opposite the closure showed no obvious adverse effects. Cell size and packing density in the shrunken IVth layer columns seemed normal. In one normal monkey in which an eye was injected the day after birth, autoradiographs of the cortex at 1 week indicated only a very mild degree of segregation of input from the two eyes; this had the form of parallel bands. Tangential recordings in layer IV C at 8 days likewise showed considerable overlap of inputs, though some segregation was clearly present; at 30 days the segregation was much more advanced. These preliminary experiments thus suggest that the layer IV C columns are not fully developed until some weeks after birth. Two alternate possibilities are considered to account for the changes in the ocular dominance columns in layer IVG following deprivation. If one ignores the above evidence in the newborn and assumes that the columns are fully formed at birth, then after eye closure the afferents from the normal eye must extend their territory, invading the deprived-eye columns perhaps by a process of sprouting of terminals. On the other hand, if at birth the fibres from each eye indeed occupy all of layer IV C, retracting to form the columns only during the first 6 weeks or so, perhaps by a process of competition, then closure of one eye may result in a competitive disadvantage of the terminals from that eye, so that they retract more than they would normally. This second possibility has the advantage that it explains the critical period for deprivation effects in the layer IV columns, this being the time after birth during which retraction is completed. It would also explain the greater severity of the changes in the earlier closures, and would provide an interpretation of both cortical and geniculate effects in terms of competition of terminals in layer IV C for territory on postsynaptic cells.

OBJECTIVE: To investigate whether placebo effects can experimentally be separated into the response to three components-assessment and observation, a therapeutic ritual (placebo treatment), and a supportive patient-practitioner relationship-and then progressively combined to produce incremental clinical improvement in patients with irritable bowel syndrome. To assess the relative magnitude of these components. DESIGN: A six week single blind three arm randomised controlled trial. SETTING: Academic medical centre. PARTICIPANTS: 262 adults (76% women), mean (SD) age 39 (14), diagnosed by Rome II criteria for and with a score of > or =150 on the symptom severity scale. INTERVENTIONS: For three weeks either waiting list (observation), placebo acupuncture alone ("limited"), or placebo acupuncture with a patient-practitioner relationship augmented by warmth, attention, and confidence ("augmented"). At three weeks, half of the patients were randomly assigned to continue in their originally assigned group for an additional three weeks. MAIN OUTCOME MEASURES: Global improvement scale (range 1-7), adequate relief of symptoms, symptom severity score, and quality of life. RESULTS: At three weeks, scores on the global improvement scale were 3.8 (SD 1.0) v 4.3 (SD 1.4) v 5.0 (SD 1.3) for waiting list versus "limited" versus "augmented," respectively (P<0.001 for trend). The proportion of patients reporting adequate relief showed a similar pattern: 28% on waiting list, 44% in limited group, and 62% in augmented group (P<0.001 for trend). The same trend in response existed in symptom severity score (30 (63) v 42 (67) v 82 (89), P<0.001) and quality of life (3.6 (8.1) v 4.1 (9.4) v 9.3 (14.0), P<0.001). All pairwise comparisons between augmented and limited patient-practitioner relationship were significant: global improvement scale (P<0.001), adequate relief of symptoms (P<0.001), symptom severity score (P=0.007), quality of life (P=0.01). Results were similar at six week follow-up. CONCLUSION: Factors contributing to the placebo effect can be progressively combined in a manner resembling a graded dose escalation of component parts. Non-specific effects can produce statistically and clinically significant outcomes and the patient-practitioner relationship is the most robust component. TRIAL REGISTRATION: Clinical Trials NCT00065403.

Highly active antiretroviral therapy has decreased human immunodeficiency virus (HIV)-associated mortality; other comorbidities, such as chronic liver disease, are assuming greater importance. We retrospectively examined the causes of death of HIV-seropositive patients at our institution in 1991, 1996, and 1998-1999. In 1998-1999, 11 (50%) of 22 deaths were due to end-stage liver disease, compared with 3 (11.5%) of 26 in 1991 and 5 (13.9%) of 36 in 1996 (P=.003). In 1998-1999, 55% of patients had nondetectable plasma HIV RNA levels and/or CD4 cell counts of >200 cells/mm(3) within the year before death. Most of the patients that were tested had detectable antibodies to hepatitis C virus (75% of patients who died in 1991, 57.7% who died in 1996, and 93.8% who died in 1998-1999; P=NS). In 1998-1999, 7 patients (31.8%) discontinued antiretroviral therapy because of hepatotoxicity, compared with 0 in 1991 and 2 (5.6%) in 1996. End-stage liver disease is now the leading cause of death in our hospitalized HIV-seropositive population.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson’s disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy. Medulloblastoma is the most common malignant brain tumour in children; having assembled over 1,000 samples the authors report that somatic copy number aberrations are common in medulloblastoma, in particular a tandem duplication of SNCAIP, a gene associated with Parkinson’s disease, which is restricted to subgroup 4α, and translocations of PVT1, which are restricted to Group 3. Medulloblastoma is the most common malignant brain tumour in children. Four papers published in the 2 August 2012 issue of Nature use whole-genome and other sequencing techniques to produce a detailed picture of the genetics and genomics of this condition. Notable findings include the identification of recurrent mutations in genes not previously implicated in medulloblastoma, with significant genetic differences associated with the four biologically distinct subgroups and clinical outcomes in each. Potential avenues for therapy are suggested by the identification of targetable somatic copy-number alterations, including recurrent events targeting TGFβ signalling in Group 3, and NF-κB signalling in Group 4 medulloblastomas.

Interoperability in healthcare has traditionally been focused around data exchange between business entities, for example, different hospital systems. However, there has been a recent push towards patient-driven interoperability, in which health data exchange is patient-mediated and patient-driven. Patient-centered interoperability, however, brings with it new challenges and requirements around security and privacy, technology, incentives, and governance that must be addressed for this type of data sharing to succeed at scale. In this paper, we look at how blockchain technology might facilitate this transition through five mechanisms: (1) digital access rules, (2) data aggregation, (3) data liquidity, (4) patient identity, and (5) data immutability. We then look at barriers to blockchain-enabled patient-driven interoperability, specifically clinical data transaction volume, privacy and security, patient engagement, and incentives. We conclude by noting that while patient-driving interoperability is an exciting trend in healthcare, given these challenges, it remains to be seen whether blockchain can facilitate the transition from institution-centric to patient-centric data sharing.

BACKGROUND: Familial hypertrophic cardiomyopathy is characterized by a variable degree of myocardial hypertrophy and a wide range of symptoms. Different mutations in the beta cardiac myosin heavy-chain gene have been identified in three affected families. However, neither the proportion of cases attributable to myosin mutations nor the effects of different mutations on clinical outcome are known. METHODS: Using a ribonuclease protection assay, we screened the beta cardiac myosin heavy-chain genes of probands from 25 unrelated families with familial hypertrophic cardiomyopathy; this assay is a sensitive method for detecting the presence and location of mutations. We further defined the mutations by analyzing their nucleotide sequences. The clinical features of the disease were compared in families with various myosin mutations. RESULTS: Seven mutations in the beta cardiac myosin heavy-chain gene were identified in 12 of the 25 families. All were missense mutations (i.e., causing the substitution of a single amino acid) clustered in the head and head-rod junction regions of the molecule. Six mutations resulted in a change in the charge of the amino acid. Patients with mutations that changed the charge of the altered amino acid (such as that from arginine to glutamine at nucleotide 403 or from arginine to cysteine at nucleotide 453) had a significantly shorter life expectancy (mean age at death, 33 years), whereas patients with the one mutation that did not produce a change in charge (Val606Met) had nearly normal survival. However, patients with different mutations did not differ appreciably in their clinical manifestations of familial hypertrophic cardiomyopathy. CONCLUSIONS: Different missense mutations in the beta cardiac myosin heavy-chain gene can be identified in approximately 50 percent of families with hypertrophic cardiomyopathy. In those families, a definite genetic diagnosis can be made in all members. Since the location of a mutation or its DNA-sequence alteration (or both) appears to influence survival, we suggest that the precise definition of the disease-causing mutation can provide important prognostic information about affected members.

Prolongation of the action potential has been postulated to be a major reason for the altered diastolic relaxation of the heart in patients with severe heart failure. To investigate the electrophysiological basis for this action potential prolongation in terminal heart failure, K+ currents were recorded in single ventricular myocytes isolated from 16 explanted hearts of patients undergoing transplantation. Results from diseased hearts were compared with ventricular cells isolated from six undiseased donor hearts. Action potential duration was significantly prolonged in cells from patients with heart failure. A delayed rectifier K+ current was hardly detectable in most cells, and if it could be recorded, it was very small in both diseased and undiseased cells. When currents were normalized for cell surface area, the average current density of the inward rectifier K+ current was significantly reduced in diseased cells when compared with normal control cells (hyperpolarization at -100 mV, -15.9 +/- 2.2 vs -9.0 +/- 1.2 microA/cm2; P < .01). In addition, a large transient outward K+ current could be recorded in human myocytes. The average current density of the time-dependent component of this transient outward K+ current was significantly reduced in heart failure (depolarization at +40 mV, 9.1 +/- 1.0 vs 5.8 +/- 0.64 microA/cm2; P < .01). Action potential prolongation in severe heart failure may partially be explained by a reduction in current densities of the inward rectifier K+ current and of the transient outward K+ current. These alterations may thereby have a significant effect on cardiac relaxation.

1. Cells in area 17 of the cat visual cortex were studied with a view towards correlating receptive field properties with layering. A number of receptive field parameters were measured for all units, and nearly every unit was marked with a microlesion to determine accurately the layer in which it was found.2. Cells were defined as simple or complex by mapping with stationary stimuli, using the criteria of Hubel & Wiesel (1962). Complex cells fell into two groups: those that showed summation for increased slit length (standard complex) and those that did not (special complex).3. The simple cells were located in the deep part of layer 3, in layer 4, and in layer 6. This corresponds to the distribution of afferents from the dorsal layers of the lateral geniculate nucleus. In these cortical layers the simple cells differed primarily with respect to their receptive field size, cells in layer 4 having the smallest, layer 3 intermediate, and layer 6 the largest fields. Layer 4 was the only layer in which simple cells showed end-inhibition (a reduction in response to slits extending beyond the excitatory portion of the receptive field).4. The standard complex cells were found in all layers, but were quite scarce in layer 4. As with the simple cells, field size varied with layer: in layer 2+3 they had small to intermediate field sizes, in layer 5 intermediate, and in layer 6 very large. Layer 6 cells showed summation for slits of increased length up to very large values, and responded best when the slits were centred in the receptive field. The only standard complex cells that showed end-inhibition were those in layer 2+3, and these were similar to the layer 4 simple cells in terms of proportion of end-inhibited units and degree of end-inhibition.5. The special complex cells, originally described by Palmer & Rosenquist (1974), were found in two tiers: the upper one at the layer 3/layer 4 border and the lower one in layer 5. They were different from the standard complex cells in having a high spontaneous activity, high velocity preference, and large fields which were similar in size (at a given eccentricity) from one cell to the next. Many showed reduced response to slits of increasing length, even for slits that did not extend beyond the borders of the responsive region.6. Cells in layer 6 (the origin of the corticogeniculate projection) were antidromically activated from the lateral geniculate nucleus. The antidromically activated units included both simple and complex cells, and they had the long receptive fields characteristic of the overall population of cells in layer 6.7. The results showed that there are different types of simple and complex cells, and that cells in different layers have different properties. Taken together with their differences in site of projection, this demonstrates that the anatomical lamination pattern is reflected in functional differences between cells in different layers.

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Seventy cases of carcinoma of the parathyroid collected over a 40-year period are analyzed. The average age of the patients was 44.3 years, and there was no sex predominance. The most common presenting signs and symptoms were bone disease, a palpable neck mass, and renal stones. The initial serum calcium value averaged 15.2 mg/100 ml. The most important histologic features were mitoses and fibrous bands. Local recurrence occurred in 30% of the cases. Metastatic spread, which occurred in 30% of the cases, was usually late and to regional lymph nodes, lungs, liver, and bone. Less than half the patients died of the disease within 5 years.

Alzheimer's disease (AD) care requires timely diagnosis and multidisciplinary management. Evaluation involves structured patient and caregiver history and symptom-function reviews, examination, and testing (laboratory and neuroimaging) to delineate impairment level, determine the cognitive-behavioral syndrome, and diagnose cause. Clinical biomarkers are available to aid high confidence in etiologic diagnosis. Management uses psychoeducation, shared goal setting, and patient-caregiver dyad decision making. When combined, pharmacologic and nonpharmacologic therapies mitigate symptoms and reduce clinical progression and care burden. AD biopathologic processes develop over decades before symptoms manifest; this period is increasingly targeted in research as an opportunity to best delay or prevent AD dementia.

Elevated plasma lipopolysaccharide (LPS), an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD). To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL) assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4(+) T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP) levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV) co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.

Ion-exchange calcium electrodes represent the first practical method for the direct measurement of ionized calcium [Ca(++)] in biologic fluids. Using both "static" and "flow-through" electrodes, serum [Ca(++)] was within a rather narrow range: 0.94-1.33 mmoles/liter (mean, 1.14 mmoles/liter). Within a given individual, [Ca(++)] varied only about 6% over a several month period. Consistent pH effects on [Ca(++)] were observed in serum and whole blood, [Ca(++)] varying inversely with pH. Less consistent pH effects were also noted in ultrafiltrates, believed to largely represent precipitation of certain calcium complexes from a supersaturated solution. Heparinized whole blood [Ca(++)] was significantly less than in corresponding serum at normal blood pH, related to the formation of a calcium-heparin complex. [Ca(++)] in ultrafiltrates represented a variable fraction (66.7-90.2%) of total diffusible calcium. There was no apparent correlation between serum ionized and total calcium concentrations. Thus, neither serum total calcium nor total ultrafiltrable calcium provided a reliable index of serum [Ca(++)]. Change in serum total calcium was almost totally accounted for by corresponding change in protein-bound calcium [CaProt]. About 81% of [CaProt] was estimated to be bound to albumin and about 19% to globulins. From observed pH, serum protein, and [CaProt] data, a nomogram was developed for estimating [CaProt] without ultrafiltration. Data presented elsewhere indicate that calcium binding by serum proteins obeys the mass-law equation for a monoligand association. This was indicated in the present studies by a close correspondence of observed serum [Ca(++)] values with those predicted by the McLean-Hastings nomogram. While these electrodes allow study of numerous problems not possible previously, they have not been perfected to the same degree of reliability obtainable with current pH electrodes. The commercial (Orion flow-through) electrode is: (a) expensive. (b) requires periodic replacement of membranes, and (c) has not yet been thermostated. As with blood pH measurements. (d) electrode response is logarithmic, i.e. small potential errors generate rather large [Ca(++)] errors. (e) loss of CO(2) should be prevented, and (f) errors due to other cations must be considered under certain conditions. Despite these limitations, we believe the electrode represents a major advance in calcium metabolism.

BACKGROUND: Since the original publication of the VCF and SAM formats, an explosion of software tools have been created to process these data files. To facilitate this a library was produced out of the original SAMtools implementation, with a focus on performance and robustness. The file formats themselves have become international standards under the jurisdiction of the Global Alliance for Genomics and Health. FINDINGS: We present a software library for providing programmatic access to sequencing alignment and variant formats. It was born out of the widely used SAMtools and BCFtools applications. Considerable improvements have been made to the original code plus many new features including newer access protocols, the addition of the CRAM file format, better indexing and iterators, and better use of threading. CONCLUSION: Since the original Samtools release, performance has been considerably improved, with a BAM read-write loop running 5 times faster and BAM to SAM conversion 13 times faster (both using 16 threads, compared to Samtools 0.1.19). Widespread adoption has seen HTSlib downloaded >1 million times from GitHub and conda. The C library has been used directly by an estimated 900 GitHub projects and has been incorporated into Perl, Python, Rust, and R, significantly expanding the number of uses via other languages. HTSlib is open source and is freely available from htslib.org under MIT/BSD license.

MICA and MICB are expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through natural killer group 2D (NKG2D) receptor activation. However, tumors evade this immune recognition pathway through proteolytic shedding of MICA and MICB proteins. We rationally designed antibodies targeting the MICA α3 domain, the site of proteolytic shedding, and found that these antibodies prevented loss of cell surface MICA and MICB by human cancer cells. These antibodies inhibited tumor growth in multiple fully immunocompetent mouse models and reduced human melanoma metastases in a humanized mouse model. Antitumor immunity was mediated mainly by natural killer (NK) cells through activation of NKG2D and CD16 Fc receptors. This approach prevents the loss of important immunostimulatory ligands by human cancers and reactivates antitumor immunity.