The SSS revolution in fungal diagnostics: speed, simplicity and sensitivity.

INTRODUCTION: Fungal disease has historically presented a diagnostic challenge due to its often non-specific clinical presentations, relative infrequency and reliance on insensitive and time-intensive fungal culture. SOURCES OF DATA: We present the recent developments in fungal diagnostics in the fields of serological and molecular diagnosis for the most clinically relevant pathogens; developments that have the potential to revolutionize fungal diagnosis through improvements in speed, simplicity and sensitivity. We have drawn on a body of evidence including recent studies and reviews demonstrating the effectiveness of antigen and antibody detection and polymerase chain reaction (PCR) in patients with and without concurrent human immunodeficiency virus infection. AREAS OF AGREEMENT: This includes recently developed fungal lateral flow assays, which have a low cost and operator skill requirement that give them great applicability to low-resource settings. Antigen detection for Cryptococcus, Histoplasma and Aspergillus spp. are much more sensitive than culture. PCR for Candida spp., Aspergillus spp., Mucorales and Pneumocystis jirovecii is more sensitive than culture and usually faster. AREAS OF CONTROVERSY: Effort must be made to utilize recent developments in fungal diagnostics in clinical settings outside of specialist centres and integrate their use into standard medical practice. Given the clinical similarities of the conditions and frequent co-infection, further study is required into the use of serological and molecular fungal tests, particularly in patients being treated for tuberculosis. GROWING POINTS: Further study is needed to clarify the utility of these tests in low-resource settings confounded by a high prevalence of tuberculosis. AREAS TIMELY FOR DEVELOPING RESEARCH: The diagnostic utility of these tests may require revision of laboratory work flows, care pathways and clinical and lab coordination, especially for any facility caring for the immunosuppressed, critically ill or those with chronic chest conditions, in whom fungal disease is common and underappreciated.

From a Collapse-Prone, Insulating Ni-MOF-74 Analogue to Crystalline, Porous, and Electrically Conducting PEDOT@MOF Composites.

Electrically conductive porous metal-organic frameworks (MOFs) show great promise in helping advance electronics and clean energy technologies. However, large porosity usually hinders long-range charge transport, an essential criterion of electrical conductivity, underscoring the need for new strategies to combine these two opposing features and realize their diverse potentials. All previous strategies to boost the conductivity of porous MOFs by introducing redox-complementary guest molecules, conducting polymers, and metal nanoparticles have led to a significant loss of frameworks' porosity and surface areas, which could be otherwise exploited to capture additional guests in electrocatalysis and chemiresistive sensing applications. Herein, we demonstrate for the first time that the in situ oxidative polymerization of preloaded 3,4-ethylenedioxythiophene (EDOT) monomers into the polyethylenedioxythiophene (PEDOT) polymer inside the hexagonal cavities of an intrinsically insulating Ni2(NDISA) MOF-74 analogue (NDISA = naphthalenediimide N,N-disalicylate), which easily collapses and becomes amorphous upon drying, simultaneously enhanced the crystallinity, porosity, and electrical conductivity of the resulting PEDOT@Ni2(NDISA) composites. At lower PEDOT loading (∼22 wt %), not only did the Brunauer-Emmett-Teller surface area of the PEDOT@Ni2(NDISA) composite (926 m2/g) more than double from that of evacuated pristine Ni2(NDISA) (387 m2/g), but also its electrical conductivity (1.1 × 10-5 S/cm) soared 105 times from that of the pristine MOF, demonstrating unprecedented dual benefits of our strategy. At higher PEDOT loading (≥33 wt %), the electrical conductivity of Ni2(NDISA)⊃PEDOT composites further increased modestly (10-4 S/cm), but their porosity dropped precipitously as large amounts of PEDOT filled up the hexagonal MOF channels. Thus, our work presents a simple new strategy to simultaneously boost the structural stability, porosity, and electrical conductivity of intrinsically insulating and collapse-prone MOFs by introducing small amounts of conducting polymers that can not only reinforce the MOF scaffolds and prevent them from collapsing but also help create a much coveted non-native property by providing charge carriers and charge transport pathways.

ppe51 Variants Enable Growth of Mycobacterium tuberculosis at Acidic pH by Selectively Promoting Glycerol Uptake.

In defined media supplemented with single carbon sources, Mycobacterium tuberculosis (Mtb) exhibits carbon source specific growth restriction. When supplied with glycerol as the sole carbon source at pH 5.7, Mtb establishes a metabolically active state of nonreplicating persistence known as acid growth arrest. We hypothesized that acid growth arrest on glycerol is not a metabolic restriction, but rather an adaptive response. To test this hypothesis, we selected for and identified several Mtb mutants that could grow under these restrictive conditions. All mutations were mapped to the ppe51 gene and resulted in variants with 3 different amino acid substitutions- S211R, E215K, and A228D. Expression of the ppe51 variants in Mtb promoted growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, Enhanced Acid Growth (EAG) phenotype. Testing growth on other single carbon sources showed the PPE51 variants specifically enhanced growth on glycerol, suggesting PPE51 plays a role in glycerol uptake. Using radiolabeled glycerol, enhanced glycerol uptake was observed in Mtb expressing the PPE51 (S211R) variant, with glycerol overaccumulation in triacylglycerol. Notably, the EAG phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication and reduced survival in activated macrophages compared to resting macrophages. Recombinant PPE51 protein exhibited differential thermostability in the wild type (WT) or S211R variants in the presence of glycerol, supporting the model that EAG substitutions alter PPE51-glycerol interactions. Together, these findings support that PPE51 variants selectively promote glycerol uptake and that slowed growth at acidic pH is an important adaptive mechanism required for macrophage pathogenesis. IMPORTANCE It is puzzling why Mycobacterium tuberculosis (Mtb) cannot grow on glycerol at acidic pH, as it has a carbon source and oxygen, everything it needs to grow. In this study, we found that Mtb limits uptake of glycerol at acidic pH to restrict its growth and that mutations in ppe51 promote uptake of glycerol at acidic pH and enable growth. That is, Mtb can grow well at acidic pH on glycerol, but has adapted instead to stop growth. Notably, ppe51 variants exhibit enhanced replication and reduced survival in activated macrophages, supporting a role for pH-dependent slowed growth during macrophage pathogenesis.

Slow growth of Mycobacterium tuberculosis at acidic pH is regulated by phoPR and host-associated carbon sources.

During pathogenesis, Mycobacterium tuberculosis (Mtb) colonizes environments, such as the macrophage or necrotic granuloma, that are acidic and rich in cholesterol and fatty acids. The goal of this study was to examine how acidic pH and available carbon sources interact to regulate Mtb physiology. Here we report that Mtb growth at acidic pH requires host-associated carbon sources that function at the intersection of glycolysis and the TCA cycle, such as pyruvate, acetate, oxaloacetate and cholesterol. In contrast, in other tested carbon sources, Mtb fully arrests its growth at acidic pH and establishes a state of non-replicating persistence. Growth-arrested Mtb is resuscitated by the addition of pyruvate suggesting that growth arrest is due to a pH-dependent checkpoint on metabolism. Additionally, we demonstrate that the phoPR two-component regulatory system is required to slow Mtb growth at acidic pH and functions to maintain redox homeostasis. Transcriptional profiling and functional metabolic studies demonstrate that signals from acidic pH and carbon source are integrated to remodel pathways associated with anaplerotic central metabolism, lipid anabolism and the regeneration of oxidized cofactors. Because phoPR is required for Mtb virulence in animals, we suggest that pH-driven adaptation may be critical to Mtb pathogenesis.

Wntless functions in mature osteoblasts to regulate bone mass.

Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are associated significantly with reduced bone mineral density. Wls/Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls-deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.

Endothelial cell stimulation overcomes restriction and promotes productive and latent HIV-1 infection of resting CD4+ T cells.

Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4(+) T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4(+) T cells, and latent infection in those cells may arise when infected CD4(+) T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4(+) T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells in vivo. Our study proposes a new mechanism for infection of resting CD4(+) T cells in vivo and a new mechanism for latent infection in resting CD4(+) T cells.

Arming Hospital Ships of the Future: Hybrid Wars Require a Major Change.

INTRODUCTION: It is time to provide heavier defense systems to U.S. Navy hospital ships. They serve vital functions in both the military and emergency management spaces. They provide medical support for combat operations and can also convey the empathy and generosity of the American people when used in humanitarian assistance and disaster relief response. Hospital ships are often key to success in scenarios that require the international deployment of resources and medical expertise. Hospital ships serve a dual purpose and hence are subject to regulations that do not address all wartime mission requirements and necessary defensive capabilities. The current U.S. Navy's interpretation of the Geneva Conventions regarding the visibility, lack of defensive capabilities, and inability to use encrypted communications needlessly endangers medical platforms and personnel in the modern environment. METHODS: The authors (including senior author F.M.B.-a recognized (International Health Law expert) reviewed relevant literature and have evaluated the policies of belligerent parties in past and current conflicts. These increasingly appear to target civilian infrastructure including medical facilities and may increase the risk to hospital ships. This demonstrable current hybrid warfare appears to include purposeful attacks on health care facilities and as such hospital ships should have additional defensive measures. RESULTS: Hybrid warfare and its focus on civilian infrastructure and health care targets are highly visible in the acts of both state and non-state actors and may encourage others to purposefully target health care facilities and personnel. Evidence of this is seen in the current Russian invasion of Ukraine, where since the invasion a year ago 1,218 Ukrainian health facilities have been damaged, including 540 damaged hospitals, 173 of which were totally destroyed and turned into "piles of stones." CONCLUSIONS: In today's conflicted global environment, the clear identification of hospital ships leaving them relatively undefended and denying encrypted communication is the folly of a bygone era. Hospital ships may be targeted because they are brightly lit soft targets that can deliver a large payoff by their destruction. It is time to adapt to the global reality and move on from the tradition of painting hospital ships white, adorning them with red crosses, keeping them unarmed, maintaining open communications, and illuminating them at night. The increasing threats from hybrid warfare and unprincipled adversaries to medical platforms and providers of health care demonstrate that hospital ships must be capable of self-defense. The U.S. Navy is designing new platforms for medical missions and the debate, no matter how uncomfortable, must now occur among major decision-makers to make them more tactical and defensible.

Intraspecies warfare restricts strain coexistence in human skin microbiomes.

Determining why only a fraction of encountered or applied bacterial strains engraft in a given person's microbiome is crucial for understanding and engineering these communities1. Previous work has established that metabolism can determine colonization success in vivo2-4, but relevance of bacterial warfare in preventing engraftment has been less explored. Here, we demonstrate that intraspecies warfare presents a significant barrier to strain transmission in the skin microbiome by profiling 14,884 pairwise interactions between Staphylococcus epidermidis cultured from eighteen human subjects from six families. We find that intraspecies antagonisms are abundant; these interactions are mechanistically diverse, independent of the relatedness between strains, and consistent with rapid evolution via horizontal gene transfer. Ability to antagonize more strains is associated with reaching a higher fraction of the on-person S. epidermidis community. Moreover, antagonisms are significantly depleted among strains residing on the same person relative to random assemblages. Two notable exceptions, in which bacteria evolved to become sensitive to antimicrobials found on the same host, are explained by mutations that provide phage resistance, contextualizing the importance of warfare among other lethal selective pressures. Taken together, our results emphasize that accounting for intraspecies bacterial warfare is essential to the design of long-lasting probiotic therapeutics.

Highly-resolved within-species dynamics in the human facial skin microbiome.

Human facial skin microbiomes (FSMs) on adults are dominated by just two bacterial species, Cutibacterium acnes and Staphylococcus epidermidis. Underlying this apparent simplicity, each FSM harbors multiple strains of both species whose assembly dynamics on individuals are unknown. Here, we use 4,055 isolate genomes and 360 metagenomes to trace the dynamics of strains on individuals and their transmission. Strains are shared amongst family members of all ages, but each individual harbors unique strain consortia. Strain stability changes upon formation of the adult-type FSM: S. epidermidis lineage turnover slows, and the rate of C. acnes colonization increases before stabilizing, suggesting this transitional window could facilitate engraftment of therapeutic strains. Our work reveals previously undetectable community dynamics and informs the design of therapeutic interventions.

Russia's Hybrid Warfare in Ukraine Threatens Both Healthcare & Health Protections Provided by International Law.

Hybrid Warfare is on display because of the unjustified Russian invasion of Ukraine. This is characterized by numerous crimes against civilians as seen vividly during the occupation of the town of Bucha where rape, torture, murder, and looting seem to reflect Russian military policy, leadership, and command guidance. Of particular concern is the threat to hospitals and health care as well as vital life support. Numerous hospitals have been damaged and destroyed. Hospitals are not tactical military targets and targeting health care facilities and personnel ignores traditional jus in bello and ignores numerous conventions established to stabilize the global order. The Russian-proclaimed "special operation" in Ukraine has been characterized by barbarian warfare in which the Russian military uses weapons against the civilian population and civilian infrastructure. The aggressors have embarked on a purposeful terror campaign through infrastructure attacks, which are of little military value except to demoralize the nation's people. This is evident with Russian missile and drone attacks on electric, water, and health care in Ukraine. Warfare now and in the future may be increasingly aimed at demoralizing civilian populations and reducing the will of the people and their government to resist. The Ukrainian invasion clearly shows that this use of hybrid warfare should be met with a strong reaction of the international community at the earliest possible stage, especially the supposedly peace-loving neutral countries, or else the future is expanded unlawful and barbaric military conflict.

Effective Humanitarian Work: Teaching Medical Skill Sets in Ukraine.

The senior authors traveled to Ukraine to teach specific skills to Ukrainian physicians and other medical professionals, utilizing a 2-day ATLS course, workshops in point-of-care ultrasonography (POCUS), lectures and webinars on damage control resuscitation, damage control surgery, and transfusion of whole blood. The authors have focused on providing skill sets that Ukrainian doctors can utilize within their existing system to improve immediate patient care for casualties resulting from the unanticipated Russian invasion and improve outcomes. Given the resource limitations and differences of the Ukrainian healthcare systems, the authors believe Western-based professionals who come to Ukraine to help for short periods should resist the temptation to offer western solutions that may not work in Ukraine. Major improvements in Ukrainian health care will require long-term efforts in teaching but also need to include increased efforts to improve hospitals, clinics, staffing, education, supplies, and equipment. Those who travel to help in Ukraine can still teach short courses that provide skills that Ukrainian doctors and nurses can use within their existing healthcare system to improve the quality of patient care in the immediate period of crisis and hopefully improve outcomes in the near term. It is not a reasonable expectation to think that the delivery of 2-day courses such as ATLS or POCUS will significantly change the country-wide delivery of healthcare. This sort of practice change requires the engagement of medical and political leaders and a sustained reform effort over years, not days or weeks. Supportive countries and non-governmental organizations need to prepare for a long and extensive investment in improving Ukrainian healthcare.

Diabetes-Associated Hyperglycemia Causes Rapid-Onset Ocular Surface Damage.

Purpose: The metabolic alterations due to chronic hyperglycemia are well-known to cause diabetes-associated complications. Short-term hyperglycemia has also been shown to cause many acute changes, including hemodynamic alterations and osmotic, oxidative, and inflammatory stress. The present study was designed to investigate whether diabetes-associated hyperglycemia can cause rapid-onset detrimental effects on the tear film, goblet cells, and glycocalyx and can lead to activation of an inflammatory cascade or cellular stress response in the cornea. Methods: Mouse models of type 1 and type 2 diabetes were used. Tear film volume, goblet cell number, and corneal glycocalyx area were measured on days 7, 14, and 28 after the onset of hyperglycemia. Transcriptome analysis was performed to quantify changes in 248 transcripts of genes involved in inflammatory, apoptotic, and stress response pathways. Results: Our data demonstrate that type 1 and type 2 diabetes-associated hyperglycemia caused a significant decrease in the tear film volume, goblet cell number, and corneal glycocalyx area. The decrease in tear film and goblet cell number was noted as early as 7 days after onset of hyperglycemia. The severity of ocular surface injury was significantly more in type 1 compared to type 2 diabetes. Diabetes mellitus also caused an increase in transcripts of genes involved in the inflammatory, apoptotic, and cellular stress response pathways. Conclusions: The results of the present study demonstrate that diabetes-associated hyperglycemia causes rapid-onset damage to the ocular surface. Thus, short-term hyperglycemia in patients with diabetes mellitus may also play an important role in causing ocular surface injury and dry eye.

Fluoroquinolone-resistant latent tuberculosis infection: A literature review and case series of 5 patients treated with linezolid monotherapy.

Latent tuberculosis infection (LTBI) constitutes an important public health problem because of risk of progression to TB disease. Effective treatment of multi-drug resistant (MDR) LTBI would prevent progression to MDR TB disease, which would improve patient and public health outcomes. The majority of MDR LTBI treatment studies have focused on the use of fluoroquinolone-based antibiotic regimens. Options for and experience in the treatment of fluoroquinolone-resistant MDR LTBI are limited in the published literature and not comprehensively addressed in current guidelines. In this review, we share our experience with the treatment of fluoroquinolone-resistant MDR LTBI with linezolid. We discuss treatment options for MDR TB that provide context for predicting effective MDR LTBI treatment, with a focus on the microbiologic and pharmacokinetic properties of linezolid that support its use. We then summarize the evidence for treatment of MDR LTBI. Finally, we present our experiences treating fluoroquinolone-resistant MDR LTBI with linezolid with an emphasis on dosing considerations to optimize efficacy and minimize potential toxicities.

Rapid, sensitive detection of PFOA with smartphone-based flow rate analysis utilizing competitive molecular interactions during capillary action.

Perfluorinated-alkyl substances (PFAS) pose an unmet threat to the public because they are not strictly monitored and regulated. Perfluorinated-carbon alkyl chains (PFOA), a type of PFAS, at 70 fg/µL is the current health and safety recommendation. Current testing methods for PFOA and PFAS chemicals include HPLC-MS/MS and molecularly imprinted polymers, which are expensive, time-consuming, and require training. In this work, PFOA and PFOS detection was performed on a paper microfluidic chip using competitive interactions between PFOA/PFOS, cellulose fibers, and various reagents (L-lysine, casein, and albumin). Such interactions altered the surface tension at the wetting front and, subsequently, the capillary flow rate. A smartphone captured the videos of this capillary action. The samples flowed through the channel in less than 2 min. Albumin worked the best in detecting PFOA, followed by casein. The detection limit was 10 ag/µL in DI water and 1 fg/µL in effluent (processed) wastewater. Specificity to other non-fluorocarbon surfactants was also tested, using anionic sodium dodecyl sulfate (SDS), non-ionic Tween 20, and cationic cetrimonium bromide (CTAB). A combination of the reagents successfully distinguished PFOA from all three surfactants at 100% accuracy. This low-cost, handheld assay can be an accessible alternative for rapid in situ estimation of PFOA concentration.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

Lrp5 and Lrp6 play compensatory roles in mouse intestinal development.

Low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) are co-receptors of Wnt ligands and play important roles in Wnt/ß-catenin signal transduction. Mice homozygous for a germline deletion of Lrp6 die at birth with several associated defects, while Lrp5-deficient mice are viable. Here, we conditionally deleted Lrp5 and/or Lrp6 in the mouse gut ((gut-/-)) by crossing mice carrying floxed alleles of Lrp5 and Lrp6 to a strain expressing Cre recombinase from the villin promoter (villin-Cre). The changes in morphology, differentiation, and Wnt signal transduction were validated using immunohistochemistry and other staining. Consistent with observations in mice carrying a homozygous germline deletion in Lrp5, intestinal development in Lrp5(gut-/-) mice was normal. In addition, mice homozygous for villin-Cre-induced deletion of Lrp6 (Lrp6(gut-/-)) were viable with apparently normal intestinal differentiation and function. However, mice homozygous for villin-Cre inactivated alleles of both genes (Lrp5(gut-/-) ; Lrp6(gut-/-)) died within 1 day of birth. Analysis of embryonic Lrp5(gut-/-); Lrp6(gut-/-) intestinal epithelium showed a progressive loss of cells, an absence of proliferation, and a premature differentiation of crypt stem/precursor cells; no notable change in differentiation was observed in the embryos lacking either gene alone. Further immunohistochemical studies showed that expression of the Wnt/ß-catenin target, cyclin D1, was specifically reduced in the intestinal epithelium of Lrp5(gut-/-); Lrp6(gut-/-) embryos. Our data demonstrate that Lrp5 and Lrp6 play redundant roles in intestinal epithelium development, and that Lrp5/6 might regulate intestinal stem/precursor cell maintenance by regulating Wnt/ß-catenin signaling.

Evidence for Vpr-dependent HIV-1 replication in human CD4+ CEM.NKR T-cells.

BACKGROUND: Vpr is exclusively expressed in primate lentiviruses and contributes to viral replication and disease progression in vivo. HIV-1 Vpr has two major activities in vitro: arrest of cell cycle in the G2 phase (G2 arrest), and enhancement of viral replication in macrophages. Previously, we reported a potent HIV-1 restriction in the human CD4+ CEM.NKR (NKR) T cells, where wild-type (WT) HIV-1 replication was inhibited by almost 1,000-fold. From the parental NKR cells, we isolated eight clones by limiting dilution. These clones showed three levels of resistance to the WT HIV-1 infection: non-permissive (NP), semi-permissive (SP), and permissive (P). Here, we compared the replication of WT, Vif-defective, Vpr-defective, and Vpu-defective viruses in these cells. RESULTS: Although both WT and Vpu-defective viruses could replicate in the permissive and semi-permissive clones, the replication of Vif-defective and Vpr-defective viruses was completely restricted. The expression of APOBEC3G (A3G) cytidine deaminase in NKR cells explains why Vif, but not Vpr, was required for HIV-1 replication. When the Vpr-defective virus life cycle was compared with the WT virus life cycle in the semi-permissive cells, it was found that the Vpr-defective virus could enter the cell and produce virions containing properly processed Gag and Env proteins, but these virions showed much less efficiency for reverse transcription during the next-round of infection. In addition, although viral replication was restricted in the non-permissive cells, treatment with arsenic trioxide (As2O3) could completely restore WT, but not Vpr-defective virus replication. Moreover, disruption of Vpr binding to its cofactor DCAF1 and/or induction of G2 arrest activity did not disrupt the Vpr activity in enhancing HIV-1 replication in NKR cells. CONCLUSIONS: These results demonstrate that HIV-1 replication in NKR cells is Vpr-dependent. Vpr promotes HIV-1 replication from the 2nd cycle likely by overcoming a block at early stage of viral replication; and this activity does not require DCAF1 and G2 arrest. Further studies of this mechanism should provide new understanding of Vpr function in the HIV-1 life cycle.

The Hospital Ship as a Strategic Asset in21st Century Foreign Policy and Global Health Crises.

INTRODUCTION: Current U.S. hospital ships-USNS Mercy and Comfort-are old, slow, cumbersome, and indefensible, and due for retirement. As new challenges and new threats emerge in the 21st century, the U.S. Navy should field new afloat medical platforms to potentially deal with both mass casualty scenarios and humanitarian disaster relief in a rapid and tactical manner. New hospital ships should be able to defend themselves with more modern weapons and to be interconnected with encrypted communications. They must be fast, nimble, tactical, defensible, and forward deployed in the risky global commons of the 21st century. MATERIALS AND METHODS: Systematic review of the literature on hospital ships, U.S. Navy policy, the Geneva Conventions, and current global threat conditions. RESULTS: Hospital ships provide medical support for U.S. forces in conflict and promote goodwill and a positive image of the U.S. abroad. Current hospital ships do not fit the operational paradigm of the current needs for forward deployed and rapidly deliverable operational medicine. There is a need for a new and more capable platform to deliver operational health care in the forward deployed setting. CONCLUSIONS: Multiple high-speed medical response vessels-whether reconfigured from an existing ship, or an entirely new platform developed for more robust medical delivery-need to be urgently fielded for future combat operations, humanitarian missions, and participation in cooperative security engagements. These medical platforms need to be able to defend themselves and be tactically interconnected with the Fleet and Fleet Forces.

Improving Pandemic Response With Military Tools: Using Enhanced Intelligence, Surveillance, and Reconnaissance.

The coronavirus disease 2019 (COVID-19) pandemic rocked the world, spurring the collapse of national commerce, international trade, education, air travel, and tourism. The global economy has been brought to its knees by the rapid spread of infection, resulting in widespread illness and many deaths. The rise in nationalism and isolationism, ethnic strife, disingenuous governmental reporting, lockdowns, travel restrictions, and vaccination misinformation have caused further problems. This has brought into stark relief the need for improved disease surveillance and health protection measures. National and international agencies that should have provided earlier warning in fact failed to do so. A robust global health network that includes enhanced cooperation with Military Intelligence, Surveillance, and Reconnaissance (ISR) assets in conjunction with the existing international, governmental, and nongovernment medical intelligence networks and allies and partners would provide exceptional forward-looking and early-warning and is a proactive step toward making our future safe. This will be achieved both by surveilling populations for new biothreats, fusing and disseminating data, and then reaching out to target assistance to reduce disease spread in unprotected populations.

Anatomy promotes neutral coexistence of strains in the human skin microbiome.

What enables strains of the same species to coexist in a microbiome? Here, we investigate whether host anatomy can explain strain co-residence of Cutibacterium acnes, the most abundant species on human skin. We reconstruct on-person evolution and migration using whole-genome sequencing of C. acnes colonies acquired from healthy subjects, including from individual skin pores, and find considerable spatial structure at the level of pores. Although lineages (sets of colonies separated by <100 mutations) with in vitro fitness differences coexist within centimeter-scale regions, each pore is dominated by a single lineage. Moreover, colonies from a pore typically have identical genomes. An absence of adaptive signatures suggests a genotype-independent source of low within-pore diversity. We therefore propose that pore anatomy imposes random single-cell bottlenecks; the resulting population fragmentation reduces competition and promotes coexistence. Our findings suggest that therapeutic interventions involving pore-dwelling species might focus on removing resident populations over optimizing probiotic fitness.