Gene Therapy with Etranacogene Dezaparvovec for Hemophilia B.

BACKGROUND: Moderate-to-severe hemophilia B is treated with lifelong, continuous coagulation factor IX replacement to prevent bleeding. Gene therapy for hemophilia B aims to establish sustained factor IX activity, thereby protecting against bleeding without burdensome factor IX replacement. METHODS: In this open-label, phase 3 study, after a lead-in period (≥6 months) of factor IX prophylaxis, we administered one infusion of adeno-associated virus 5 (AAV5) vector expressing the Padua factor IX variant (etranacogene dezaparvovec; 2×1013 genome copies per kilogram of body weight) to 54 men with hemophilia B (factor IX activity ≤2% of the normal value) regardless of preexisting AAV5 neutralizing antibodies. The primary end point was the annualized bleeding rate, evaluated in a noninferiority analysis comparing the rate during months 7 through 18 after etranacogene dezaparvovec treatment with the rate during the lead-in period. Noninferiority of etranacogene dezaparvovec was defined as an upper limit of the two-sided 95% Wald confidence interval of the annualized bleeding rate ratio that was less than the noninferiority margin of 1.8. Superiority, additional efficacy measures, and safety were also assessed. RESULTS: The annualized bleeding rate decreased from 4.19 (95% confidence interval [CI], 3.22 to 5.45) during the lead-in period to 1.51 (95% CI, 0.81 to 2.82) during months 7 through 18 after treatment, for a rate ratio of 0.36 (95% Wald CI, 0.20 to 0.64; P<0.001), demonstrating noninferiority and superiority of etranacogene dezaparvovec as compared with factor IX prophylaxis. Factor IX activity had increased from baseline by a least-squares mean of 36.2 percentage points (95% CI, 31.4 to 41.0) at 6 months and 34.3 percentage points (95% CI, 29.5 to 39.1) at 18 months after treatment, and usage of factor IX concentrate decreased by a mean of 248,825 IU per year per participant in the post-treatment period (P<0.001 for all three comparisons). Benefits and safety were observed in participants with predose AAV5 neutralizing antibody titers of less than 700. No treatment-related serious adverse events occurred. CONCLUSIONS: Etranacogene dezaparvovec gene therapy was superior to prophylactic factor IX with respect to the annualized bleeding rate, and it had a favorable safety profile. (Funded by uniQure and CSL Behring; HOPE-B ClinicalTrials.gov number, NCT03569891.).

Progenitor identification and SARS-CoV-2 infection in human distal lung organoids.

The distal lung contains terminal bronchioles and alveoli that facilitate gas exchange. Three-dimensional in vitro human distal lung culture systems would strongly facilitate the investigation of pathologies such as interstitial lung disease, cancer and coronavirus disease 2019 (COVID-19) pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we describe the development of a long-term feeder-free, chemically defined culture system for distal lung progenitors as organoids derived from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids were able to differentiate into AT1 cells, and basal cell organoids developed lumens lined with differentiated club and ciliated cells. Single-cell analysis of KRT5+ cells in basal organoids revealed a distinct population of ITGA6+ITGB4+ mitotic cells, whose offspring further segregated into a TNFRSF12Ahi subfraction that comprised about ten per cent of KRT5+ basal cells. This subpopulation formed clusters within terminal bronchioles and exhibited enriched clonogenic organoid growth activity. We created distal lung organoids with apical-out polarity to present ACE2 on the exposed external surface, facilitating infection of AT2 and basal cultures with SARS-CoV-2 and identifying club cells as a target population. This long-term, feeder-free culture of human distal lung organoids, coupled with single-cell analysis, identifies functional heterogeneity among basal cells and establishes a facile in vitro organoid model of human distal lung infections, including COVID-19-associated pneumonia.

A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production.

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

The dissection of a despotic society: exploration, dominance and hormonal traits.

Naked mole-rats (Heterocephalus glaber) live in large colonies with one breeding female (queen), one to three breeding males (BMs) and the remainder are non-reproductive subordinates. The animals have a linear dominance rank with the breeders at the top of the hierarchy. We investigated how dominance rank in naked mole-rats differs with exploration (the propensity to explore a novel environment) and related endocrine markers. Exploration behaviour, faecal progestagen metabolite (fPM), faecal glucocorticoid metabolite (fGCM), faecal androgen metabolite (fAM) and plasma prolactin concentrations were quantified in breeding, high-, middle- and low-ranked females and males from five naked mole-rat colonies. There were no significant differences between the dominance rank and exploration behaviour. Interestingly, the queens and high-ranking females had higher fGCM and fAM concentrations compared with middle- and low-ranked females. The queens had significantly higher fPM concentrations than all other ranked females, since they are responsible for procreation. In the males, the BMs had higher fGCM concentrations compared with high- and low-ranked males. In addition, BMs and middle-ranking males had overall higher prolactin levels than all other ranked males, which could be linked to cooperative care. Overall, the results suggest that physiological reproductive suppression is linked to high dominance rank.

The Relationship Between Transfusion in Cardiac Surgery Patients and Adverse Outcomes.

OBJECTIVES: To understand if red blood cell (RBC) transfusions are independently associated with a risk of mortality, prolonged intubation, or infectious, cardiac, or renal morbid outcomes. DESIGN: A retrospective review. SETTING: A single-institution university hospital. PARTICIPANTS: A total of 2,458 patients undergoing coronary bypass artery graft and/or valvular surgery from July 2014 through January 2018. INTERVENTIONS: No interventions were done. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the occurrence of an adverse event or prolonged intubation. Infectious, cardiac, and renal composite outcomes were also defined. These composites, along with mortality, were analyzed individually and then combined to form the "any adverse events" composite. Preoperative demographic and intraoperative parameters were analyzed as univariate risk factors for adverse outcomes. Logistic regression was used to screen variables, with a p value criterion of p < 0.05 for entry into the model selection procedure. A backward selection algorithm was used with variable entry and retention criteria of p < 0.05 to select the final multivariate model. Multivariate logistic regression models were used to determine whether there was an association between the volume of RBC transfusion and the defined adverse event after adjusting for covariates. A p value < 0.01 was considered statistically significant in the final model of each aim to adjust for multiple comparisons. The final logistic models for each of the following outcomes indicate an increased risk of that outcome per each additional unit of RBC transfused. For prolonged intubation, the odds ratio (OR) was 1.493 (p < 0.0001), OR = 1.358 (p < 0.0001) for infectious composite outcomes, OR = 1.247 (p < 0.0001) for adverse renal outcomes, and OR = 1.467 (p < 0.0001) for any adverse event. CONCLUSIONS: The authors demonstrated a strong independent association between RBC transfusion volume and adverse outcomes after cardiac surgery. Efforts should be undertaken, such as preoperative anemia management and control of coagulopathy, in order to minimize the need for RBC transfusion.

A cohort study and matched pair analysis evaluating the effects of the COVID-19 pandemic on access to dental care for people with inherited bleeding disorders.

INTRODUCTION: Patients with inherited bleeding disorders (IBDs) can experience challenges in accessing dental care. The COVID-19 pandemic saw the cessation of routine dentistry in England. This study aims to highlight whether access to dental care for patients with IBDs was impacted by the pandemic, and whether the severity of their IBD impacts patients experience of dental care. METHODS: Patients over the age of 18 with Haemophilia A and B, Von Willibrands (VWD), Bernard-Soulier syndrome (BSS) and Glanzmann's disease were recruited. One hundred and seventy-eight patients were called and invited to complete a questionnaire. Full cohort and matched pair analysis was performed. RESULTS: Seventy participants completed the questionnaire (62 males, mean age 39.9), 26 had a mild IBD and 44 severe. Mild and severe patients were matched on IBD, gender, deprivation index and age. Thirty-one percent of patients tried to seek dental advice during the pandemic. Eighty-one percent of mild patients saw a dentist routinely prior to the pandemic, compared to 58% of severe patients. Doctors were more likely to discuss the importance of dental prevention with patients with severe IBDs (p = .0042). CONCLUSION: Fewer patients with severe IBDs accessed regular dental care compared to their matched pair counterparts prior to the pandemic. This could indicate that access to care is more difficult for those with severe IBDs. Patients felt that oral health is relevant to their IBD. While doctors were more likely to discuss the importance of preventing dental disease with patients who have severe IBDs, the profession needs to ensure this translates into routine engagement.

Emicizumab prophylaxis in haemophilia A with inhibitors: Three years follow-up from the UK Haemophilia Centre Doctors' Organisation (UKHCDO).

INTRODUCTION: The UK National Haemophilia Database (NHD) collects data from all UK persons with haemophilia A with inhibitors (PwHA-I). It is well-placed to investigate patient selection, clinical outcomes, drug safety and other issues not addressed in clinical trials of emicizumab. AIMS: To determine safety, bleeding outcomes and early effects on joint health of emicizumab prophylaxis in a large, unselected cohort using national registry and patient reported Haemtrack (HT) data between 01 January 2018 and 30 September 2021. METHODS: Prospectively collected bleeding outcomes were analysed in people with ≥6 months emicizumab HT data and compared with previous treatment if available. Change in paired Haemophilia Joint Health Scores (HJHS) were analysed in a subgroup. Adverse events (AEs) reports were collected and adjudicated centrally. RESULTS: This analysis includes 117 PwHA-I. Mean annualised bleeding rate (ABR) was .32 (95% CI, .18; .39) over a median 42 months treatment with emicizumab. Within-person comparison (n = 74) demonstrated an 89% reduction in ABR after switching to emicizumab and an increase in zero treated bleed rate from 45 to 88% (p < .01). In a subgroup of 37 people, total HJHS improved in 36%, remained stable in 46% and deteriorated in 18%, with a median (IQR) within-person change of -2.0 (-9, 1.5) (p = .04). Three arterial thrombotic events were reported, two possibly drug related. Other AEs were generally non-severe and usually limited to early treatment, included cutaneous reactions (3.6%), headaches (1.4%), nausea (2.8%) and arthralgia (1.4%). CONCLUSIONS: Emicizumab prophylaxis is associated with sustained low bleeding rates and was generally well-tolerated in people with haemophilia A and inhibitors.

The basal transcription complex component TAF3 transduces changes in nuclear phosphoinositides into transcriptional output.

Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers.

A comprehensive profile of reproductive hormones in eusocial Damaraland mole-rats (Fukomys damarensis).

In species where sociality and group cohesion are primarily determined by the maintenance of a reproductive division of labour and cooperative behaviours, the eusocial Damaraland mole-rat (Fukomys damarensis) presents a model which provides behavioural and endocrine distinctions between sex (males and females) and reproductive class (breeders and non-breeders). Although previous studies have demonstrated the endocrine aspects of reproductive suppression and behaviour in Damaraland mole-rats, they have focused on one hormone separately and on different conspecifics and samples across time. Unfortunately, this could introduce extrinsic biases when using these studies to compile complete hormonal profiles for comparisons. This study, therefore, set out to obtain a profile of the reproductive hormones from breeding and non-breeding male and female Damaraland mole-rats at a single point in time, from which circulating plasma prolactin and urinary progesterone, testosterone, and cortisol were measured. As expected, plasma prolactin and urinary cortisol did not differ between the breeders and non-breeders. However, breeders (both male and female) possessed increased urinary testosterone and progesterone concentrations compared to their non-breeding counterparts. These results, in conjunction with the variation in the expression of the respective hormonal receptors within the brains of breeders and non-breeders suggest that elevated testosterone and progesterone in breeders establish a neural dominance phenotype, which ultimately aids in controlling breeding activities. This study has emphasised the need for holistic, comprehensive profiling of reproductive endocrine systems.

The relationship between hypoxia exposure and circulating cortisol levels in social and solitary African mole-rats: An initial report.

Hypoxemia from exposure to intermittent and/or acute environmental hypoxia (lower oxygen concentration) is a severe stressor for many animal species. The response to hypoxia of the hypothalamic-pituitary-adrenal axis (HPA-axis), which culminates in the release of glucocorticoids, has been well-studied in hypoxia-intolerant surface-dwelling mammals. Several group-living (social) subterranean species, including most African mole-rats, are hypoxia-tolerant, likely due to regular exposure to intermittent hypoxia in their underground burrows. Conversely, solitary mole-rat species, lack many adaptive mechanisms, making them less hypoxia-tolerant than the social genera. To date, the release of glucocorticoids in response to hypoxia has not been measured in hypoxia-tolerant mammalian species. Consequently, this study exposed three social African mole-rat species and two solitary mole-rat species to normoxia, or acute hypoxia and then measured their respective plasma glucocorticoid (cortisol) concentrations. Social mole-rats had lower plasma cortisol concentrations under normoxia than the solitary genera. Furthermore, individuals of all three of the social mole-rat species exhibited significantly increased plasma cortisol concentrations after hypoxia, similar to those of hypoxia-intolerant surface-dwelling species. By contrast, individuals of the two solitary species had a reduced plasma cortisol response to acute hypoxia, possibly due to increased plasma cortisol under normoxia. If placed in perspective with other closely related surface-dwelling species, the regular exposure of the social African mole-rats to hypoxia may have reduced the basal levels of the components for the adaptive mechanisms associated with hypoxia exposure, including circulating cortisol levels. Similarly, the influence of body mass on plasma cortisol levels cannot be ignored. This study demonstrates that both hypoxia-tolerant rodents and hypoxia-intolerant terrestrial laboratory-bred rodents may possess similar HPA-axis responses from exposure to hypoxia. Further research is required to confirm the results from this pilot study and to further confirm how the cortisol concentrations may influence responses to hypoxia in African mole-rats.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Delivery of AAV-based gene therapy through haemophilia centres-A need for re-evaluation of infrastructure and comprehensive care: A Joint publication of EAHAD and EHC.

INTRODUCTION: Adeno-associated virus (AAV)-based gene therapy for haemophilia presents a challenge to the existing structure of haemophilia centres and requires a rethink of current collaboration and information exchange with the aim of ensuring a system that is fit-for-purpose for advanced therapies to maximise benefits and minimise risks. In Europe, a certification process based on the number of patients and facilities is offered to the haemophilia centres by European Haemophilia Network (EUHANET). AIM AND METHODS: This joint European Association for Haemophilia and Allied Disorders (EAHAD) and European Haemophilia Consortium (EHC) publication describes criteria for centres participating in gene therapy care that require a reassessment of the infrastructure of comprehensive care and provides an outlook on how these criteria can be implemented in the future work of haemophilia centres. RESULTS: The core definition of a haemophilia treatment centre remains, but additional roles could be implemented. A modifiable 'hub-and-spoke' model addresses all aspects associated with gene therapy, including preparation and administration of the gene therapy product, determination of coagulation and immunological parameters, joint score and function, and liver health. This will also include the strategy on how to follow-up patients for a long-term safety and efficacy surveillance. CONCLUSION: We propose a modifiable, networked 'hub and spoke' model with a long term safety and efficacy surveillance system. This approach will be progressively developed with the goal of making haemophilia centres better qualified to deliver gene therapy and to make gene therapy accessible to all persons with haemophilia, irrespective of their country or centre of origin.

Factor VIII/IX inhibitor testing practices in the United Kingdom: Results of a UKHCDO and UKNEQAS national survey.

INTRODUCTION: Inhibitor formation is the greatest challenge facing persons with haemophilia treated with factor concentrates. The gold standard testing methodologies are the Nijmegen-Bethesda assay (NBA) for FVIII and Bethesda assay (BA) for FIX inhibitors, which are affected by pre-analytical and inter-laboratory variability. AIMS: To evaluate inhibitor testing methodology and assess correlation between self-reported and actual methodology. METHODS: Methodology was evaluated using a survey distributed alongside a UK National External Quality Assessment Service Blood Coagulation external quality assurance (EQA) exercise for FVIII and FIX inhibitor testing. RESULTS: Seventy four survey and EQA exercise responses were received (response rate 63.2%), with 50 paired survey/EQA results. 47.1% (33/70) reported using the NBA and 42.9% (30/70) the BA for FVIII inhibitor testing. Review of FVIII inhibitor assay methodology demonstrated discrepancy (self-reported to actual) in 64.3% (BA reporting) and 27.6% (NBA reporting). Pre-analytical heat treatment was used by 32.4%, most commonly 56°C for 30 minutes. Assay cut-offs of 0.1-1.0 BU/mL were reported. EQA samples (acquired FVIII and congenital FIX) demonstrated titres and coefficients of variation (CV) of 3.1 BU/mL (0.7-15.4 BU/mL; CV = 43%) and 18.0 BU/mL (0-117 BU/mL; CV = 33%), respectively. No significant assay or laboratory factors were found to explain this variance, which could have resulted in change in management for 6 patients (5 misclassified high-titre FVIII inhibitors and 1 false negative for a FIX inhibitor). CONCLUSIONS: Heterogeneity was seen at each stage of assay methodology. No assay-related factors were found to explain variation in inhibitor titres. Further standardization is required to improve inhibitor quantification to guide patient care.

Immune tolerance induction in severe haemophilia A: A UKHCDO inhibitor and paediatric working party consensus update.

INTRODUCTION: In good risk patients (historic inhibitor peak < 200BU), the International Immune Tolerance Study demonstrated equal efficacy to induce tolerance between high (200iu/kg/day) and low dose (50iu/kg ×3 times/week) immune tolerance induction (ITI) regimens. However, the trial stopped early on account of the excessive bleed rate in the low dose ITI arm. METHODS: United Kingdom Haemophilia Centre Doctors' Organization (UKHCDO) Paediatric and Inhibitor working parties considered available ITI data alongside the bi-phenotypic antibody emicizumab (Hemlibra®) efficacy and safety data to develop a consensus guideline for the future UK ITI guideline. RESULTS: This revision of UKHCDO ITI guidance incorporates the recommendation to use emicizumab as a prophylaxis haemostatic agent to reduce bleeding rates and to facilitate low dose and reduced frequency of FVIII CFC for ITI in the majority of children. CONCLUSION: This consensus protocol will facilitate future evaluation of ITI outcomes in the evolving landscape of haemophilia therapeutics and ITI strategies.

PERSEPT 3: A phase 3 clinical trial to evaluate the haemostatic efficacy of eptacog beta (recombinant human FVIIa) in perioperative care in subjects with haemophilia A or B with inhibitors.

INTRODUCTION: Surgical procedures in persons with haemophilia A or B with inhibitors (PwHABI) require the use of bypassing agents (BPA) and carry a high risk of complications. Historically, only two BPAs have been available; these are reported to have variable responses. AIM: To prospectively evaluate the efficacy and safety of a new bypassing agent, human recombinant factor VIIa (eptacog beta) in elective surgical procedures in PwHABI in a phase 3 clinical trial, PERSEPT 3. METHODS: Subjects were administered 200 µg/kg (major procedures) or 75 µg/kg eptacog beta (minor procedures) immediately prior to the initial surgical incision; subsequent 75 µg/kg doses were administered to achieve postoperative haemostasis and wound healing. Efficacy was assessed on a 4-point haemostatic scale during the intra- and postoperative periods. Anti-drug antibodies, thrombotic events and changes in clinical/laboratory parameters were monitored throughout the perioperative period. RESULTS: Twelve subjects underwent six major and six minor procedures. The primary efficacy endpoint success proportion was 100% (95% CI: 47.8%-100%) for minor procedures and 66.7% (95% CI: 22.3%-95.7%) for major procedures; 81.8% (95% CI: 48.2%-97.7%) of the procedures were considered successful using eptacog beta. There was one death due to bleeding from a nonsurgical site; this was assessed as unlikely related to eptacog beta. No thrombotic events or anti-eptacog beta antibodies were reported. CONCLUSION: Two eptacog beta dosing regimens in PwHABI undergoing major and minor surgical procedures were well-tolerated, and the majority of procedures were successful based on surgeon/investigator assessments. Eptacog beta offers clinicians a new potential therapeutic option for procedures in PwHABI.

Plasma oxidative stress in reproduction of two eusocial African mole-rat species, the naked mole-rat and the Damaraland mole-rat.

One of the most prominent life-history trade-offs involves the cost of reproduction. Oxidative stress has been proposed to be involved in this trade-off and has been associated with reduced life span. There is currently an unclear relationship between oxidative cost and the reproduction-longevity trade-off. The current study, using a non-lethal and minimally invasive (only a single blood sample and no euthanasia) method, investigated whether an oxidative cost (oxidative stress) to reproduction would be apparent in two long-lived eusocial mole-rats, the naked mole-rat (NMR), Heterocephalus glaber, and the Damaraland mole-rat (DMR), Fukomys damarensis, where breeding colony members live longer than non-breeder conspecifics. We measured the direct redox balance in plasma by measuring the oxidative stress index (OSI) based on the ratio of total oxidant status and total antioxidant activity in breeders and non-breeders of both sexes, in the two species. NMR had significantly higher OSI between breeders and non-breeders of each sex, whereas DMR showed no significant differences except for total antioxidant capacity (TAC). The mode of reproductive suppression and the degree of reproductive investment in NMR may explain to some degree the redox balance difference between breeders and non-breeders. DMR show minimal physiological changes between breeders and non-breeders except for the mode of reproduction, which may explain some variations in TAC and TOS values, but similar OSI between breeders and non-breeders.

The joint effect of micro- and macro-climate on the thermoregulation and heat dissipation of two African mole-rat (Bathyergidae) sub-species, Cryptomys hottentotus mahali and C. h. pretoriae.

The effect of the macro- and microclimate on small mammal thermoregulation in the past has been studied independently instead of investigating the dual effect of both the components. This study addresses this dearth in knowledge by exploring the dual effect of both micro- and macro-climate on the thermoregulatory responses of two subterranean rodent species belonging to the family Bathyergidae, namely the more arid dwelling Mahali mole-rat (Cryptomys hottentotus mahali) and less arid dwelling Highveld mole-rat (C. h. pretoriae). Open flow through respirometry was used to quantify resting metabolic rates (RMR), evaporative water loss (EWL), core body temperature (Tb), the ratio between the evaporative heat loss and metabolic heat production (EHL/MHP: evaporative cooling capacity) and conductance (Cdry) over a range of increasing ambient temperatures (Ta; 20-42 °C). Furthermore, RMR, EWL, Tb, EHL/MHP and Cdry were measured at the mole-rat's thermal maxima (43 °C). At cooler temperatures, the arid-dwelling C. h. mahali possesses a broader thermoneutral zone (~5 °C; 27.2-32.1 °C), while C. h. pretoriae possess a single thermoneutral point (33.6 °C). This is in response to the greater selection pressure to conserve energy in the more arid regions inhabited by C. h. mahali. Contrastingly, at hotter temperatures, there were no significant thermoregulatory differences in EWL, EHL/MHP or Cdry responses between the two sub-species, as expected due to the limitations bestowed by the buffered microclimates (burrow systems). Thus, neither macro-climate, nor micro-climate singularly moulds the thermoregulatory adaptations, but rather, it appears to be a combined effect from both climates. Other small endotherms may share this dual response, and therefore, it is crucial to incorporate the effect of both macro- and microclimates into future climate models when determining the ecological capabilities and persistence of a species.

The brains of six African mole-rat species show divergent responses to hypoxia.

Mole-rats are champions of self-preservation, with increased longevity compared with other rodents their size, strong antioxidant capabilities and specialized defenses against endogenous oxidative stress. However, how the brains of these subterranean mammals handle acute in vivo hypoxia is poorly understood. This study is the first to examine the molecular response to low oxygen in six different species of hypoxia-tolerant mole-rats from sub-Saharan Africa. Protein carbonylation, a known marker of DNA damage (hydroxy-2'-deoxyguanosine), and antioxidant capacity did not change following hypoxia but HIF-1 protein levels increased significantly in the brains of two species. Nearly 30 miRNAs known to play roles in hypoxia tolerance were differentially regulated in a species-specific manner. The miRNAs exhibiting the strongest response to low oxygen stress inhibit apoptosis and regulate neuroinflammation, likely providing neuroprotection. A principal component analysis (PCA) using a subset of the molecular targets assessed herein revealed differences between control and hypoxic groups for two solitary species (Georychus capensis and Bathyergus suillus), which are ecologically adapted to a normoxic environment, suggesting a heightened sensitivity to hypoxia relative to species that may experience hypoxia more regularly in nature. By contrast, all molecular data were included in the PCA to detect a difference between control and hypoxic populations of eusocial Heterocephalus glaber, indicating they may require many lower-fold changes in signaling pathways to adapt to low oxygen settings. Finally, none of the Cryptomys hottentotus subspecies showed a statistical difference between control and hypoxic groups, presumably due to hypoxia tolerance derived from environmental pressures associated with a subterranean and social lifestyle.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia.

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

Factor VIII cross-matches to the human proteome reduce the predicted inhibitor risk in missense mutation hemophilia A.

Single missense mutations in the F8 gene encoding the coagulation protein factor VIII give rise predominantly to non-severe hemophilia A. Despite only a single amino acid sequence difference between the replacement, therapeutic factor VIII and the patient's endogenous factor VIII, therapeutic factor VIII may still be perceived as foreign by the recipient's immune system and trigger an immune response (inhibitor). Inhibitor formation is a life-long risk for patients with non-severe hemophilia A treated with therapeutic factor VIII, but remains difficult to predict. The aim of this study was to understand whether fortuitous, primary sequence cross-matches between therapeutic factor VIII and proteins in the human proteome are the reason why certain F8 mutations are not associated with inhibitor formation. We predicted which therapeutic factor VIII differences are potentially perceived as foreign by helper T cells - a necessary precursor to inhibitor development - and then scanned potentially immunogenic peptides against more than 100,000 proteins in the proteome. As there are hundreds of disease-causing F8 missense mutations and the human leukocyte antigen gene complex governing peptide presentation to helper T cells is highly polymorphic, these calculations pose a huge combinatorial challenge that we addressed computationally. We found that cross-matches between therapeutic factor VIII and the human proteome are commonplace and have a profound impact on the predicted risk of inhibitor development. Our results emphasize the importance of knowing both the F8 missense mutation and the human leukocyte antigen alleles of a patient with missense mutation hemophilia A if his underlying risk of inhibitor development is to be estimated.