The impact of inversions across 33,924 families with rare disease from a national genome sequencing project.

Detection of structural variants (SVs) is currently biased toward those that alter copy number. The relative contribution of inversions toward genetic disease is unclear. In this study, we analyzed genome sequencing data for 33,924 families with rare disease from the 100,000 Genomes Project. From a database hosting >500 million SVs, we focused on 351 genes where haploinsufficiency is a confirmed disease mechanism and identified 47 ultra-rare rearrangements that included an inversion (24 bp to 36.4 Mb, 20/47 de novo). Validation utilized a number of orthogonal approaches, including retrospective exome analysis. RNA-seq data supported the respective diagnoses for six participants. Phenotypic blending was apparent in four probands. Diagnostic odysseys were a common theme (>50 years for one individual), and targeted analysis for the specific gene had already been performed for 30% of these individuals but with no findings. We provide formal confirmation of a European founder origin for an intragenic MSH2 inversion. For two individuals with complex SVs involving the MECP2 mutational hotspot, ambiguous SV structures were resolved using long-read sequencing, influencing clinical interpretation. A de novo inversion of HOXD11-13 was uncovered in a family with Kantaputra-type mesomelic dysplasia. Lastly, a complex translocation disrupting APC and involving nine rearranged segments confirmed a clinical diagnosis for three family members and resolved a conundrum for a sibling with a single polyp. Overall, inversions play a small but notable role in rare disease, likely explaining the etiology in around 1/750 families across heterogeneous clinical cohorts.

Elevating theranostics: The emergence and promise of radiopharmaceutical cell-targeting heterodimers in human cancers.

Optimal therapeutic and diagnostic efficacy is essential for healthcare's global mission of advancing oncologic drug development. Accurate diagnosis and detection are crucial prerequisites for effective risk stratification and personalized patient care in clinical oncology. A paradigm shift is emerging with the promise of multi-receptor-targeting compounds. While existing detection and staging methods have demonstrated some success, the traditional approach of monotherapy is being reevaluated to enhance therapeutic effectiveness. Heterodimeric site-specific agents are a versatile solution by targeting two distinct biomarkers with a single theranostic agent. This review describes the innovation of dual-targeting compounds, examining their design strategies, therapeutic implications, and the promising path they present for addressing complex diseases.

Opaganib Downregulates N-Myc Expression and Suppresses In Vitro and In Vivo Growth of Neuroblastoma Cells.

Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody.

The Sphingolipid-Modulating Drug Opaganib Protects against Radiation-Induced Lung Inflammation and Fibrosis: Potential Uses as a Medical Countermeasure and in Cancer Radiotherapy.

Fibrosis is a chronic pathology resulting from excessive deposition of extracellular matrix components that leads to the loss of tissue function. Pulmonary fibrosis can follow a variety of diverse insults including ischemia, respiratory infection, or exposure to ionizing radiation. Consequently, treatments that attenuate the development of debilitating fibrosis are in desperate need across a range of conditions. Sphingolipid metabolism is a critical regulator of cell proliferation, apoptosis, autophagy, and pathologic inflammation, processes that are all involved in fibrosis. Opaganib (formerly ABC294640) is the first-in-class investigational drug targeting sphingolipid metabolism for the treatment of cancer and inflammatory diseases. Opaganib inhibits key enzymes in sphingolipid metabolism, including sphingosine kinase-2 and dihydroceramide desaturase, thereby reducing inflammation and promoting autophagy. Herein, we demonstrate in mouse models of lung damage following exposure to ionizing radiation that opaganib significantly improved long-term survival associated with reduced lung fibrosis, suppression of granulocyte infiltration, and reduced expression of IL-6 and TNFα at 180 days after radiation. These data further demonstrate that sphingolipid metabolism is a critical regulator of fibrogenesis, and specifically show that opaganib suppresses radiation-induced pulmonary inflammation and fibrosis. Because opaganib has demonstrated an excellent safety profile during clinical testing in other diseases (cancer and COVID-19), the present studies support additional clinical trials with this drug in patients at risk for pulmonary fibrosis.

Detailed Analysis of ITPR1 Missense Variants Guides Diagnostics and Therapeutic Design.

BACKGROUND: The ITPR1 gene encodes the inositol 1,4,5-trisphosphate (IP3 ) receptor type 1 (IP3 R1), a critical player in cerebellar intracellular calcium signaling. Pathogenic missense variants in ITPR1 cause congenital spinocerebellar ataxia type 29 (SCA29), Gillespie syndrome (GLSP), and severe pontine/cerebellar hypoplasia. The pathophysiological basis of the different phenotypes is poorly understood. OBJECTIVES: We aimed to identify novel SCA29 and GLSP cases to define core phenotypes, describe the spectrum of missense variation across ITPR1, standardize the ITPR1 variant nomenclature, and investigate disease progression in relation to cerebellar atrophy. METHODS: Cases were identified using next-generation sequencing through the Deciphering Developmental Disorders study, the 100,000 Genomes project, and clinical collaborations. ITPR1 alternative splicing in the human cerebellum was investigated by quantitative polymerase chain reaction. RESULTS: We report the largest, multinational case series of 46 patients with 28 unique ITPR1 missense variants. Variants clustered in functional domains of the protein, especially in the N-terminal IP3 -binding domain, the carbonic anhydrase 8 (CA8)-binding region, and the C-terminal transmembrane channel domain. Variants outside these domains were of questionable clinical significance. Standardized transcript annotation, based on our ITPR1 transcript expression data, greatly facilitated analysis. Genotype-phenotype associations were highly variable. Importantly, while cerebellar atrophy was common, cerebellar volume loss did not correlate with symptom progression. CONCLUSIONS: This dataset represents the largest cohort of patients with ITPR1 missense variants, expanding the clinical spectrum of SCA29 and GLSP. Standardized transcript annotation is essential for future reporting. Our findings will aid in diagnostic interpretation in the clinic and guide selection of variants for preclinical studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Single-cell atlas of the liver myeloid compartment before and after cure of chronic viral hepatitis.

BACKGROUND & AIMS: Chronic viral infections present serious public health challenges; however, direct-acting antivirals (DAAs) are now able to cure nearly all patients infected with hepatitis C virus (HCV), representing the only cure of a human chronic viral infection to date. DAAs provide a valuable opportunity to study immune pathways in the reversal of chronic immune failures in an in vivo human system. METHODS: To leverage this opportunity, we used plate-based single-cell RNA-seq to deeply profile myeloid cells from liver fine needle aspirates in patients with HCV before and after DAA treatment. We comprehensively characterised liver neutrophils, eosinophils, mast cells, conventional dendritic cells, plasmacytoid dendritic cells, classical monocytes, non-classical monocytes, and macrophages, and defined fine-grained subpopulations of several cell types. RESULTS: We discovered cell type-specific changes post-cure, including an increase in MCM7+STMN1+ proliferating CD1C+ conventional dendritic cells, which may support restoration from chronic exhaustion. We observed an expected downregulation of interferon-stimulated genes (ISGs) post-cure as well as an unexpected inverse relationship between pre-treatment viral load and post-cure ISG expression in each cell type, revealing a link between viral loads and sustained modifications of the host's immune system. We found an upregulation of PD-L1/L2 gene expression in ISG-high neutrophils and IDO1 expression in eosinophils, pinpointing cell subpopulations crucial for immune regulation. We identified three recurring gene programmes shared by multiple cell types, distilling core functions of the myeloid compartment. CONCLUSIONS: This comprehensive single-cell RNA-seq atlas of human liver myeloid cells in response to cure of chronic viral infections reveals principles of liver immunity and provides immunotherapeutic insights. CLINICAL TRIAL REGISTRATION: This study is registered at ClinicalTrials.gov (NCT02476617). IMPACT AND IMPLICATIONS: Chronic viral liver infections continue to be a major public health problem. Single-cell characterisation of liver immune cells during hepatitis C and post-cure provides unique insights into the architecture of liver immunity contributing to the resolution of the first curable chronic viral infection of humans. Multiple layers of innate immune regulation during chronic infections and persistent immune modifications after cure are revealed. Researchers and clinicians may leverage these findings to develop methods to optimise the post-cure environment for HCV and develop novel therapeutic approaches for other chronic viral infections.

Opaganib (ABC294640) Induces Immunogenic Tumor Cell Death and Enhances Checkpoint Antibody Therapy.

Antibody-based cancer drugs that target the checkpoint proteins CTLA-4, PD-1 and PD-L1 provide marked improvement in some patients with deadly diseases such as lung cancer and melanoma. However, most patients are either unresponsive or relapse following an initial response, underscoring the need for further improvement in immunotherapy. Certain drugs induce immunogenic cell death (ICD) in tumor cells in which the dying cells promote immunologic responses in the host that may enhance the in vivo activity of checkpoint antibodies. Sphingolipid metabolism is a key pathway in cancer biology, in which ceramides and sphingosine 1-phosphate (S1P) regulate tumor cell death, proliferation and drug resistance, as well as host inflammation and immunity. In particular, sphingosine kinases are key sites for manipulation of the ceramide/S1P balance that regulates tumor cell proliferation and sensitivity to radiation and chemotherapy. We and others have demonstrated that inhibition of sphingosine kinase-2 by the small-molecule investigational drug opaganib (formerly ABC294640) kills tumor cells and increases their sensitivities to other drugs and radiation. Because sphingolipids have been shown to regulate ICD, opaganib may induce ICD and improve the efficacy of checkpoint antibodies for cancer therapy. This was demonstrated by showing that in vitro treatment with opaganib increases the surface expression of the ICD marker calreticulin on a variety of tumor cell types. In vivo confirmation was achieved using the gold standard immunization assay in which B16 melanoma, Lewis lung carcinoma (LLC) or Neuro-2a neuroblastoma cells were treated with opaganib in vitro and then injected subcutaneously into syngeneic mice, followed by implantation of untreated tumor cells 7 days later. In all cases, immunization with opaganib-treated cells strongly suppressed the growth of subsequently injected tumor cells. Interestingly, opaganib treatment induced crossover immunity in that opaganib-treated B16 cells suppressed the growth of both untreated B16 and LLC cells and opaganib-treated LLC cells inhibited the growth of both untreated LLC and B16 cells. Next, the effects of opaganib in combination with a checkpoint antibody on tumor growth in vivo were assessed. Opaganib and anti-PD-1 antibody each slowed the growth of B16 tumors and improved mouse survival, while the combination of opaganib plus anti-PD-1 strongly suppressed tumor growth and improved survival (p < 0.0001). Individually, opaganib and anti-CTLA-4 antibody had modest effects on the growth of LLC tumors and mouse survival, whereas the combination of opaganib with anti-CTLA-4 substantially inhibited tumor growth and increased survival (p < 0.001). Finally, the survival of mice bearing B16 tumors was only marginally improved by opaganib or anti-PD-L1 antibody alone but was nearly doubled by the drugs in combination (p < 0.005). Overall, these studies demonstrate the ability of opaganib to induce ICD in tumor cells, which improves the antitumor activity of checkpoint antibodies.

Spiking neural networks fine-tuning for brain image segmentation.

Introduction: The field of machine learning has undergone a significant transformation with the progress of deep artificial neural networks (ANNs) and the growing accessibility of annotated data. ANNs usually require substantial power and memory usage to achieve optimal performance. Spiking neural networks (SNNs) have recently emerged as a low-power alternative to ANNs due to their sparsity nature. Despite their energy efficiency, SNNs are generally more difficult to be trained than ANNs. Methods: In this study, we propose a novel three-stage SNN training scheme designed specifically for segmenting human hippocampi from magnetic resonance images. Our training pipeline starts with optimizing an ANN to its maximum capacity, then employs a quick ANN-SNN conversion to initialize the corresponding spiking network. This is followed by spike-based backpropagation to fine-tune the converted SNN. In order to understand the reason behind performance decline in the converted SNNs, we conduct a set of experiments to investigate the output scaling issue. Furthermore, we explore the impact of binary and ternary representations in SNN networks and conduct an empirical evaluation of their performance through image classification and segmentation tasks. Results and discussion: By employing our hybrid training scheme, we observe significant advantages over both ANN-SNN conversion and direct SNN training solutions in terms of segmentation accuracy and training efficiency. Experimental results demonstrate the effectiveness of our model in achieving our design goals.

Little Patients, Big Tasks - A Pediatric Emergency Medicine Escape Room.

Audience: The target audience for this small group session is post-graduate year (PGY) 1-4 emergency medicine (EM) residents, pediatric EM (PEM) fellows, and medical students. Introduction: Pediatric emergency department visits have been declining since the start of the COVID-19 pandemic, leading to decreased exposure to pediatric emergency care for EM residents and other learners in the ED.1 This is a major problem, given that the Accreditation Council for Graduate Medical Education (ACGME) mandates that a minimum of 20% of patient encounters or five months of training time for EM residents must occur with pediatric patients, with at least 50% of that time spent in the ED setting.2,3 A minimum of 12 months must be spent in the pediatric ED for PEM fellows,2 and an average of 7.1 weeks of medical school are spent in pediatric clerkships.4 This decrease in pediatrics exposure in the post-pandemic environment can be addressed through simulation and gamification. We selected the gamification method of an escape room to create an engaging environment in which learners could interface with key pediatric emergency medicine clinical concepts via group learning. Educational Objectives: By the end of this small group exercise, learners will be able to:Demonstrate appropriate dosing of pediatric code and resuscitation medicationsRecognize normal pediatric vital signs by ageDemonstrate appropriate use of formulas to calculate pediatric equipment sizes and insertion depthsRecognize classic pediatric murmursAppropriately diagnose congenital cardiac conditionsRecognize abnormal pediatric electrocardiograms (ECGs)Identify life-threatening pediatric conditionsDemonstrate intraosseous line (IO) insertion on a pediatric modelDemonstrate appropriate use of the Neonatal Resuscitation Protocol (NRP®) algorithms. Educational Methods: An escape room - a form of gamification - was utilized to engage the learners in active learning. Gamification is an increasingly popular educational technique being utilized in graduate medical education and refers to the conversion of serious, non-trivial material into a fun activity fashioned like a game in order to enhance engagement in learning.5 This educational method seeks to enhance knowledge, attitudes, and skills via components of games - such as puzzles and prizes - outside of the context of a traditional game.6 Though high-quality research data on the effectiveness of gamification methods in graduate medical education is limited, studies have shown that gamification enhances learning, attitudes, and behaviors.5,7 One randomized, clinical-controlled trial investigating the use of gamification to enhance patient outcomes found that patients of primary care physicians randomized to the gamification group reached blood pressure targets faster than in the control group.8 Escape rooms as a modality for education have been suggested to improve active learning and enhance learner engagement in the learning process.9 In an escape room, learners are "locked" in an artificial environment (whether digitally or in person) and must utilize their group or individual knowledge to solve puzzles and escape from their "entrapment."9,10 Escape rooms utilized as part of EM residency didactic training have demonstrated learner enthusiasm,11,12 desire to repeat the activity again,13 preference for escape rooms over traditional learning methods,14,15 improved confidence in communication and leadership skills,11,15 and improvement scores from pre- to post-testing.16We developed an escape room in which learners were divided into teams and informed that they would need to "escape" from our resident lounge by successfully completing all nine stations. The first team to complete all nine stations would win a prize. Only after the last team completed the ninth station and debriefing was complete could all teams be "freed" from the escape room. Research Methods: Learners provided anonymous online survey feedback regarding the quality of the educational content and the efficacy of the delivery method. Results: A post-participation survey was disseminated to 55 residents, 32 of whom attended the PEM Escape Room, with a response rate of 9% (3/32 residents). One hundred percent of respondents felt that the activity content was applicable to their needs as an emergency physician. The session was rated as excellent by 33.3% of respondents, and 66.7% of respondents rated the session as above average. A second survey was disseminated seven months after the event to the 24 remaining residents who attended the event, with a response rate of 46% (11/24 residents); eight attendees had graduated at the time of this survey dissemination. Results of the second survey indicated that 100% (24/24 residents) felt that the activity content was applicable to their needs as an emergency physician, 73% (17/24 residents) rated the session as excellent, and 27% (7/24 residents) rated the session as above average. Discussion: Though we received limited survey responses (3/32 on the first survey and 11/24 on the second survey), respondents felt that the educational content met their learning needs and was of high quality. We had six faculty members present to facilitate the escape room while there were four groups of residents (eight per group). The ideal faculty to resident ratio would be one faculty member per group with three to six players, based on prior literature showing that teams of more than six players take longer to complete escape room tasks.17,18 We also recognized the importance of sending out the feedback survey link early because we believe the delay in our survey being emailed to the residents contributed to the low response rate (three trainees).One participant provided the following feedback: "I think the 'escape room' struck an excellent balance with regard to trying to address knowledge that was relevant but also obscure or difficult enough that group/collaborative effort was required. I enjoyed the process and low stakes atmosphere." This quote nicely summarizes our take-aways: That the PEM escape room incorporates key tenets of adult learning theory. Also known as andragogy, adult learning theory posits that adult learners are self-directed, have prior life experiences that shape their learning process, learn for practical reasons (ie, choose to learn in order to fulfill the demands of their social role), and are problem-oriented in their learning.19 Though andragogy does not technically apply only to adults (as many children are self-directed learners),20 having an understanding of the practical and experiential nature via which adults approach learning allows the adult educator to appropriately cater educational activities to meet the adult learner's needs.This escape room aligned with the core tenets of adult learning theory in several ways. Specifically, residents were given autonomy of participation in the escape room and thus had to take initiative to promote their own learning.21 Topics featured in the escape room stations were selected based on their clinical challenges and high-yield for board examinations and patient care, making their relevance immediately obvious to learners; this is a key feature of catering to adult learners.22 The escape room provided a comfortable and collegial environment in which residents felt comfortable learning, fostering an ideal setting for mature learners.21 Direct and immediate feedback are key components of adult learning theory, and faculty members were physically present to provide feedback at each escape room station.22 Finally, working in teams required the learners to engage in active learning rather than acting as passive recipients of cognitive information.21 Thus, the PEM escape room serves as an ideal framework to meet the needs of the adult learner. Topics: Pediatrics, emergency medicine, pediatric emergency medicine.

The Systems Measurement of Mammalian Biotas, Part Two.

For a recent publication, the authors identified a seven-region model of mammal family distribution patterns, in which each unit contributes equally to the system's overall statistical characteristics of diversity, despite its individual units having measurably different levels of diversity and endemism. This systemization presents a highly efficient descriptive model that can possibly be interpreted as a form of natural classification. An additional analysis of the same mode is described here, in which the seven-region model of the distribution of mammal families' spatial affinities is shown to closely approach a most-probable-state arrangement, as assessed through combinatorics, raising some important questions about how macroevolutionary patterns might self-organize spatially. One of the possible practical applications of the overall approach is to areal representation; statistical moments of the underlying world patterns can be used to characterize faunal statuses at any individual location by relating the latter to the former. Through this approach, classical concepts such as corridors, tracks, and transition zones might be re-examined in a manner that better lends itself to hypothesis testing. An arbitrarily chosen bounded area, the conterminous United States, is treated in this fashion by way of illustration.

Efferocytosis by bone marrow mesenchymal stromal cells disrupts osteoblastic differentiation via mitochondrial remodeling.

The efficient clearance of dead and dying cells, efferocytosis, is critical to maintain tissue homeostasis. In the bone marrow microenvironment (BMME), this role is primarily fulfilled by professional bone marrow macrophages, but recent work has shown that mesenchymal stromal cells (MSCs) act as a non-professional phagocyte within the BMME. However, little is known about the mechanism and impact of efferocytosis on MSCs and on their function. To investigate, we performed flow cytometric analysis of neutrophil uptake by ST2 cells, a murine bone marrow-derived stromal cell line, and in murine primary bone marrow-derived stromal cells. Transcriptional analysis showed that MSCs possess the necessary receptors and internal processing machinery to conduct efferocytosis, with Axl and Tyro3 serving as the main receptors, while MerTK was not expressed. Moreover, the expression of these receptors was modulated by efferocytic behavior, regardless of apoptotic target. MSCs derived from human bone marrow also demonstrated efferocytic behavior, showing that MSC efferocytosis is conserved. In all MSCs, efferocytosis impaired osteoblastic differentiation. Transcriptional analysis and functional assays identified downregulation in MSC mitochondrial function upon efferocytosis. Experimentally, efferocytosis induced mitochondrial fission in MSCs. Pharmacologic inhibition of mitochondrial fission in MSCs not only decreased efferocytic activity but also rescued osteoblastic differentiation, demonstrating that efferocytosis-mediated mitochondrial remodeling plays a critical role in regulating MSC differentiation. This work describes a novel function of MSCs as non-professional phagocytes within the BMME and demonstrates that efferocytosis by MSCs plays a key role in directing mitochondrial remodeling and MSC differentiation. Efferocytosis by MSCs may therefore be a novel mechanism of dysfunction and senescence. Since our data in human MSCs show that MSC efferocytosis is conserved, the consequences of MSC efferocytosis may impact the behavior of these cells in the human skeleton, including bone marrow remodeling and bone loss in the setting of aging, cancer and other diseases.

Opioid cutting agents for use as internal standards in ion mobility spectrometry (IMS).

Opioids have become a serious public health concern over the last decade. These compounds are commonly found mixed, or cut, with safer compounds to make the opioids appear unadulterated while also enhancing the psychoactive effect on the user. Commercial benchtop and handheld IMS devices are capable of detection but published reduced ion mobility (K0) values, used to identify the target analytes with IMS instrumentation, have shown variability. This lack of agreement, even for compounds used for calibration, is often due to the effects of drift tube temperature, drift gas water vapor levels and the use in-house built instrumentation rather than commercial equipment. Multiple reports exist on assessment of IMS reference standards but a single, consensus universal standard does not exist. Assessment of opioid cutting agents as internal standards is a worthwhile pursuit if precise and accurate K0 values are obtained. The effects of drift gas water vapor content and drift tube temperature were used to evaluate the cutting agents. The K0 values of papaverine, a representative opioid with a similar K0 value to heroin and fentanyl, were calculated with respect to quinine and were in agreement with literature data. The use of quinine as an internal standard also improved precision relative to the instrument standard and shows promise in the application presented here.

Improving carbon monoxide tolerance of Cupriavidus necator H16 through adaptive laboratory evolution.

Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.

Development of multidrug-resistant Mycobacterium tuberculosis in the biofilm of a peritoneal-venous shunt.

A patient with ascites received a peritoneal-venous shunt for presumed cirrhosis, however surgical specimens grew Mycobacterium tuberculosis (MTb) sensitive to all anti-tuberculous drugs. Directly-Observed-Therapy (DOT) led to improvement followed by relapse with multidrug resistant MTb (MDRTB). We discuss pathways for selection of MDRTB within mycobacterial biofilm. This case illustrates the potential for development of MDRTB in patients with long-term indwelling catheters. We emphasize catheter removal and if not possible continuing follow-up for symptoms and signs of relapse.

Evolution and Biogeography, and the Systems Measurement of Mammalian Biotas.

Biological evolution is generally regarded as a stochastic or probabilistic process, per the ideas of Darwin in the nineteenth century. Even if this is true at the meso-scale, it still may, however, be impacted by overarching constraints that we have not yet identified. In this paper, we revisit the subject of mammal faunal regions with a mind to explore a potential kind of macroevolutionary influence. We first identify an optimum seven-region mammal faunal classification system based on spatial and phylogenetic data from a comprehensive 2013 review, and then examine the possibility that this classification provides supporting evidence for a Spinoza-influenced philosophical/theoretical model of the "natural system" concept developed by one of the authors in the 1980s. The hierarchical pattern of regional affinities revealed does do this.

Evaluating the evidence behind umbilical cord clamping practices in at-risk neonatal populations.

Umbilical cord clamping practices impact nearly 140 million births each year. Current evidence has led professional organizations to recommend delayed cord clamping (DCC), as opposed to early cord clamping (ECC), as the standard of care in uncomplicated term and preterm deliveries. However, variability remains in cord management practices for maternal-infant dyads at higher risk of complications. This review examines the current state of evidence on the outcomes of at-risk infant populations receiving differing umbilical cord management strategies. Review of contemporary literature demonstrates members of high-risk neonatal groups, including those affected by small for gestational age (SGA) classification, intrauterine growth restriction (IUGR), maternal diabetes, and Rh-isoimmunization, are frequently excluded from participation in clinical trials of cord clamping strategies. Furthermore, when these populations are included, outcomes are often underreported. Consequently, evidence regarding optimal umbilical cord management in at-risk groups is limited, and further research is needed to guide best clinical practice.

Dentin defects caused by a Dspp-1 frameshift mutation are associated with the activation of autophagy.

Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' - 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp-1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp-1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp-1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp-1fs mice may be valuable for the study of autophagy and ER-phagy.