Role of sulfidogenic members of the gut microbiota in human disease.

The human gut flora comprises a dynamic network of bacterial species that coexist in a finely tuned equilibrium. The interaction with intestinal bacteria profoundly influences the host's development, metabolism, immunity, and overall health. Furthermore, dysbiosis, a disruption of the gut microbiota, can induce a variety of diseases, not exclusively associated with the intestinal tract. The increased consumption of animal protein, high-fat and high-sugar diets in Western countries has been implicated in the rise of chronic and inflammatory illnesses associated with dysbiosis. In particular, this diet leads to the overgrowth of sulfide-producing bacteria, known as sulfidogenic bacteria, which has been linked to inflammatory bowel diseases and colorectal cancer, among other disorders. Sulfidogenic bacteria include sulfate-reducing bacteria (Desulfovibrio spp.) and Bilophila wadsworthia among others, which convert organic and inorganic sulfur compounds to sulfide through the dissimilatory sulfite reduction pathway. At high concentrations, sulfide is cytotoxic and disrupts the integrity of the intestinal epithelium and mucus barrier, triggering inflammation. Besides producing sulfide, B. wadsworthia has revealed significant pathogenic potential, demonstrated in the ability to cause infection, adhere to intestinal cells, promote inflammation, and compromise the integrity of the colonic mucus layer. This review delves into the mechanisms by which taurine and sulfide-driven gut dysbiosis contribute to the pathogenesis of sulfidogenic bacteria, and discusses the role of these gut microbes, particularly B. wadsworthia, in human diseases.

Identification of homologs of the Chlamydia trachomatis effector CteG reveals a family of Chlamydiaceae type III secreted proteins that can be delivered into host cells.

Chlamydiae are a large group of obligate endosymbionts of eukaryotes that includes the Chlamydiaceae family, comprising several animal pathogens. Among Chlamydiaceae, Chlamydia trachomatis causes widespread ocular and urogenital infections in humans. Like many bacterial pathogens, all Chlamydiae manipulate host cells by injecting them with type III secretion effector proteins. We previously characterized the C. trachomatis effector CteG, which localizes at the host cell Golgi and plasma membrane during distinct phases of the chlamydial infectious cycle. Here, we show that CteG is a Chlamydiaceae-specific effector with over 60 homologs phylogenetically categorized into two distinct clades (CteG I and CteG II) and exhibiting several inparalogs and outparalogs. Notably, cteG I homologs are syntenic to C. trachomatis cteG, whereas cteG II homologs are syntenic among themselves but not with C. trachomatis cteG. This indicates a complex evolution of cteG homologs, which is unique among C. trachomatis effectors, marked by numerous events of gene duplication and loss. Despite relatively modest sequence conservation, nearly all tested CteG I and CteG II proteins were identified as type III secretion substrates using Yersinia as a heterologous bacterial host. Moreover, most of the type III secreted CteG I and CteG II homologs were delivered by C. trachomatis into host cells, where they localized at the Golgi region and cell periphery. Overall, this provided insights into the evolution of bacterial effectors and revealed a Chlamydiaceae family of type III secreted proteins that underwent substantial divergence during evolution while conserving the capacity to localize at specific host cell compartments.

Non-conductive silicon-containing materials improve methane production by pure cultures of methanogens.

Conductive materials (CM) enhance methanogenesis, but there is no clear correlation between conductivity and faster methane production (MP) rates. We investigated if MP by pure cultures of methanogens (Methanobacterium formicicum, Methanospirillum hungatei, Methanothrix harundinacea and Methanosarcina barkeri) is affected by CM (activated carbon (AC), magnetite), and other sustainable alternatives (sand and glass beads, without conductivity, and zeolites (Zeo)). The significant impact of the materials was on M. formicicum as MP was significantly accelerated by non-CM (e.g., sand reduced the lag phase (LP) duration by 48 %), Zeo and AC (LP reduction in 71% and 75 %, respectively). Conductivity was not correlated with LP reduction. Instead, silicon content in the materials was inversely correlated with the time required for complete MP, and silicon per se stimulated M. formicicum's activity. These findings highlight the potential of using non-CM silicon-containing materials in anaerobic digesters to accelerate methanogenesis.

Infection vs. Reinfection: The Immunomodulation of Erythropoiesis.

Severe malarial anemia (SMA) increases the morbidity and mortality of Plasmodium, the causative agent of malaria. SMA is mainly developed by children and pregnant women in response to the infection. It is characterized by ineffective erythropoiesis caused by impaired erythropoietin (EPO) signaling. To gain new insights into the pathogenesis of SMA, we investigated the relationship between the immune system and erythropoiesis, conducting comparative analyses in a mouse model of malaria. Red blood cell (RBC) production was evaluated in infected and reinfected animals to mimic endemic occurrences. Higher levels of circulating EPO were observed in response to (re)infection. Despite no major differences in bone marrow erythropoiesis, compensatory mechanisms of splenic RBC production were significantly reduced in reinfected mice. Concomitantly, a pronounced immune response activation was observed in erythropoietic organs of reinfected animals in relation to single-infected mice. Aged mice were also used to mimic the occurrence of malaria in the elderly. The increase in symptom severity was correlated with the enhanced activation of the immune system, which significantly impaired erythropoiesis. Immunocompromised mice further support the existence of an immune-shaping regulation of RBC production. Overall, our data reveal the strict correlation between erythropoiesis and immune cells, which ultimately dictates the severity of SMA.

First-trimester uterine artery Doppler and hypertensive disorders in twin pregnancies: Use of twin versus singleton references.

OBJECTIVE: To determine the association of first-trimester uterine artery Doppler with hypertensive disorders of pregnancy in twin pregnancies. METHODS: This was a retrospective cohort study of twin pregnancies followed at the University Hospital Center of Central Lisbon, Portugal, between January 2010 and December 2022. First-trimester uterine artery pulsatility index (UtA-PI) was determined and compared between twin pregnancies (n = 454) and singleton pregnancies (n = 908), matched to maternal and pregnancy characteristics. Maternal characteristics and mean UtA-PI were analyzed for gestational age, birth weight, gestational hypertension, early- and late-onset pre-eclampsia, HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome, and preterm birth. Univariable and multivariable logistic regression models were used. RESULTS: The mean first-trimester UtA-PI was significantly lower in dichorionic twins than in singletons (P < 0.001). To study hypertensive disorders of pregnancy in twins, 390 pregnancies were included: 311 (79.7%) dichorionic and 79 (20.3%) monochorionic twins. The observed rates of early- and late-onset pre-eclampsia, gestational hypertension, and HELLP syndrome were 1.0%, 4.4%, 7.4%, and 1.5%, respectively. We achieved a 100% detection rate for early-onset pre-eclampsia using the UtA-PI 90th centile for twins. However, when singleton references were considered, the detection rate decreased to 50%. UtA-PI at or above the 95th centile was associated with increased odds for preterm birth before 32 weeks (adjusted odds ratio 4.1, 95% confidence interval 1.0-16.7, P = 0.043). CONCLUSIONS: Unless other major risk factors for hypertensive disorders are present, women with low UtA-PI will probably not benefit from aspirin prophylaxis. Close monitoring of all twin pregnancies for hypertensive disorders is still recommended.

Structural and biochemical characterization of the M405S variant of Desulfovibrio vulgaris formate dehydrogenase.

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed. This variant displayed reduced activity in both formate oxidation and CO2 reduction, together with an increased sensitivity to oxygen inactivation.

First-trimester serum biomarkers in twin pregnancies and adverse obstetric outcomes-a single center cohort study.

PURPOSE: This study aimed to determine the association of first-trimester maternal serum biomarkers with preterm birth (PTB), fetal growth restriction (FGR) and hypertensive disorders of pregnancy (HDP) in twin pregnancies. METHODS: This is a retrospective cohort study of twin pregnancies followed at Maternidade Dr. Alfredo da Costa, Lisbon, Portugal, between January 2010 and December 2022. We included women who completed first-trimester screening in our unit and had ongoing pregnancies with two live fetuses, and delivered after 24 weeks. Maternal characteristics, pregnancy-associated plasma protein-A (PAPP-A) and ß-human chorionic gonadotropin (ß-hCG) levels were analyzed for different outcomes: small for gestational age (SGA), gestational hypertension (GH), early and late-onset pre-eclampsia (PE), as well as the composite outcome of PTB associated with FGR and/or HDP. Univariable, multivariable logistic regression analyses and receiver-operating characteristic curve were used. RESULTS: 466 twin pregnancies met the inclusion criteria. Overall, 185 (39.7%) pregnancies were affected by SGA < 5th percentile and/or HDP. PAPP-A demonstrated a linear association with gestational age at birth and mean birth weight. PAPP-A proved to be an independent risk factor for SGA and PTB (< 34 and < 36 weeks) related to FGR and/or HDP. None of the women with PAPP-A MoM > 90th percentile developed early-onset PE or PTB < 34 weeks. CONCLUSION: A high serum PAPP-A (> 90th percentile) ruled out early-onset PE and PTB < 34 weeks. Unless other major risk factors for hypertensive disorders are present, these women should not be considered candidates for aspirin prophylaxis. Nevertheless, close monitoring of all TwP for adverse obstetric outcomes is still recommended.

Efficacy and cost analysis of intravenous conscious sedation for long oral surgery procedures.

The aim of this study was to determine what is considered a long oral surgery and conduct a cost-effective analysis of sedative agents used for intravenous sedation (IVS) and sedation protocols for such procedures. Pubmed and Google Scholar databases were used to identify human studies employing IVS for extractions and implant-related surgeries, between 2003 and July/2023. Sedation protocols and procedure lengths were documented. Sedative satisfaction, operator satisfaction, and sedation assessment were also recorded. Cost estimation was based on The British National Formulary (BNF). To assess bias, the Cochrane Risk of Bias tools were employed. This review identified 29 randomised control trials (RCT), six cohorts, 14 case-series, and one case-control study. The study defined long procedures with an average duration of 31.33 minutes for extractions and 79.37 minutes for implant-related surgeries. Sedative agents identified were midazolam, dexmedetomidine, propofol, and remimazolam. Cost analysis revealed midazolam as the most cost-effective option (<10 pence per procedure per patient) and propofol the most expensive option (approximately £46.39). Bias analysis indicated varying degrees of bias in the included studies. Due to diverse outcome reporting, a comparative network approach was employed and revealed benefits of using dexmedetomidine, propofol, and remimazolam over midazolam. Midazolam, dexmedetomidine, propofol, and remimazolam demonstrated safety and efficacy as sedative agents for conscious IVS in extended procedures like extractions or implant-related surgeries. While midazolam is the most cost-effective option, dexmedetomidine, propofol, and remimazolam offer subjective and clinical benefits. The relatively higher cost of propofol may impede its widespread use. Dexmedetomidine and remimazolam stand out as closely priced options, necessitating further clinical investigations for comparative efficacy assessment.

Energy flux couples sulfur isotope fractionation to proteomic and metabolite profiles in Desulfovibrio vulgaris.

Microbial sulfate reduction is central to the global carbon cycle and the redox evolution of Earth's surface. Tracking the activity of sulfate reducing microorganisms over space and time relies on a nuanced understanding of stable sulfur isotope fractionation in the context of the biochemical machinery of the metabolism. Here, we link the magnitude of stable sulfur isotopic fractionation to proteomic and metabolite profiles under different cellular energetic regimes. When energy availability is limited, cell-specific sulfate respiration rates and net sulfur isotope fractionation inversely covary. Beyond net S isotope fractionation values, we also quantified shifts in protein expression, abundances and isotopic composition of intracellular S metabolites, and lipid structures and lipid/water H isotope fractionation values. These coupled approaches reveal which protein abundances shift directly as a function of energy flux, those that vary minimally, and those that may vary independent of energy flux and likely do not contribute to shifts in S-isotope fractionation. By coupling the bulk S-isotope observations with quantitative proteomics, we provide novel constraints for metabolic isotope models. Together, these results lay the foundation for more predictive metabolic fractionation models, alongside interpretations of environmental sulfur and sulfate reducer lipid-H isotope data.

Integrating genetic and immune factors to uncover pathogenetic mechanisms of viral-associated pulmonary aspergillosis.

Invasive pulmonary aspergillosis is a severe fungal infection primarily affecting immunocompromised patients. Individuals with severe viral infections have recently been identified as vulnerable to developing invasive fungal infections. Both influenza-associated pulmonary aspergillosis (IAPA) and COVID-19-associated pulmonary aspergillosis (CAPA) are linked to high mortality rates, emphasizing the urgent need for an improved understanding of disease pathogenesis to unveil new molecular targets with diagnostic and therapeutic potential. The recent establishment of animal models replicating the co-infection context has offered crucial insights into the mechanisms that underlie susceptibility to disease. However, the development and progression of human viral-fungal co-infections exhibit a significant degree of interindividual variability, even among patients with similar clinical conditions. This observation implies a significant role for host genetics, but information regarding the genetic basis for viral-fungal co-infections is currently limited. In this review, we discuss how genetic factors known to affect either antiviral or antifungal immunity could potentially reveal pathogenetic mechanisms that predispose to IAPA or CAPA and influence the overall disease course. These insights are anticipated to foster further research in both pre-clinical models and human patients, aiming to elucidate the complex pathophysiology of viral-associated pulmonary aspergillosis and contributing to the identification of new diagnostic and therapeutic targets to improve the management of these co-infections.

The secretome of macrophages has a differential impact on spinal cord injury recovery according to the polarization protocol.

Introduction: The inflammatory response after spinal cord injury (SCI) is an important contributor to secondary damage. Infiltrating macrophages can acquire a spectrum of activation states, however, the microenvironment at the SCI site favors macrophage polarization into a pro-inflammatory phenotype, which is one of the reasons why macrophage transplantation has failed. Methods: In this study, we investigated the therapeutic potential of the macrophage secretome for SCI recovery. We investigated the effect of the secretome in vitro using peripheral and CNS-derived neurons and human neural stem cells. Moreover, we perform a pre-clinical trial using a SCI compression mice model and analyzed the recovery of motor, sensory and autonomic functions. Instead of transplanting the cells, we injected the paracrine factors and extracellular vesicles that they secrete, avoiding the loss of the phenotype of the transplanted cells due to local environmental cues. Results: We demonstrated that different macrophage phenotypes have a distinct effect on neuronal growth and survival, namely, the alternative activation with IL-10 and TGF-ß1 (M(IL-10+TGF-ß1)) promotes significant axonal regeneration. We also observed that systemic injection of soluble factors and extracellular vesicles derived from M(IL-10+TGF-ß1) macrophages promotes significant functional recovery after compressive SCI and leads to higher survival of spinal cord neurons. Additionally, the M(IL-10+TGF-ß1) secretome supported the recovery of bladder function and decreased microglial activation, astrogliosis and fibrotic scar in the spinal cord. Proteomic analysis of the M(IL-10+TGF-ß1)-derived secretome identified clusters of proteins involved in axon extension, dendritic spine maintenance, cell polarity establishment, and regulation of astrocytic activation. Discussion: Overall, our results demonstrated that macrophages-derived soluble factors and extracellular vesicles might be a promising therapy for SCI with possible clinical applications.

Lingual Frenotomy in Pediatric Ankyloglossia: A Diode Laser Approach in Two Case Reports.

Ankyloglossia can be related to a number of complications, such as breastfeeding difficulties or alterations in craniofacial development. Treatment can involve surgery to correct the altered lingual frenulum and can be performed by various techniques. The purpose of this paper is to present two case reports of ankyloglossia in pediatric patients of different ages, the diagnostic criteria, and the treatment decision rationale, which led to a lingual frenotomy performed with a diode laser.

Adipose tissue derived stem cell secretome induces motor and histological gains after complete spinal cord injury in Xenopus laevis and mice.

Mesenchymal stem cell-based therapies have been studied for spinal cord injury (SCI) treatment due to their paracrine action upon damaged tissues. MSCs neuroregenerative role may relate to the contents of their secretome in anti-inflammatory cytokines and growth-permissive factors. We propose using the secretome of MSCs isolated from the adipose tissue-adipose tissue-derived stem cells (ASCs) as a cell-free based therapy for SCI. In vivo studies were conducted in two SCI models, Xenopus laevis and mice, after complete spinal cord transection. Our results on both models demonstrated positive impacts of ASC secretome on their functional recovery which were correlated with histopathological markers of regeneration. Furthermore, in our mice study, secretome induced white matter preservation together with modulation of the local and peripheral inflammatory response. Altogether, these results demonstrate the neuroregenerative and potential for inflammatory modulation of ASC secretome suggesting it as a good candidate for cell-free therapeutic strategies for SCI.

DsrMKJOP is the terminal reductase complex in anaerobic sulfate respiration.

Microbial dissimilatory sulfate reduction (DSR) is a key process in the Earth biogeochemical sulfur cycle. In spite of its importance to the sulfur and carbon cycles, industrial processes, and human health, it is still not clear how reduction of sulfate to sulfide is coupled to energy conservation. A central step in the pathway is the reduction of sulfite by the DsrAB dissimilatory sulfite reductase, which leads to the production of a DsrC-trisulfide. A membrane-bound complex, DsrMKJOP, is present in most organisms that have DsrAB and DsrC, and its involvement in energy conservation has been inferred from sequence analysis, but its precise function was so far not determined. Here, we present studies revealing that the DsrMKJOP complex of the sulfate reducer Archaeoglobus fulgidus works as a menadiol:DsrC-trisulfide oxidoreductase. Our results reveal a close interaction between the DsrC-trisulfide and the DsrMKJOP complex and show that electrons from the quinone pool reduce consecutively the DsrM hemes b, the DsrK noncubane [4Fe-4S]3+/2+ catalytic center, and finally the DsrC-trisulfide with concomitant release of sulfide. These results clarify the role of this widespread respiratory membrane complex and support the suggestion that DsrMKJOP contributes to energy conservation upon reduction of the DsrC-trisulfide in the last step of DSR.

An allosteric redox switch involved in oxygen protection in a CO2 reductase.

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

Higher risk of preterm twin delivery among shorter nulliparous women.

OBJECTIVE: To determine if maternal height in nulliparous women influences pregnancy results in twin pregnancies. MATERIAL AND METHODS: Retrospective cohort analysis evaluating twin pregnancies followed at Centro Hospitalar Universitário Lisboa Central, between 1995 and 2020. Of the 2900 pregnancies followed in that period, 886 nulliparous women with dichorionic twin pregnancies were selected. Two groups were considered: A - maternal height <163 cm (

Soft tissue expansion using self-inflating osmotic hydrogel expanders prior to bone augmentation: healing and complications. Evidence-based review.

AIM: This review aims to assess complication rates, soft tissue gain, and bone gain associated with the use of self-inflating osmotic hydrogel tissue expanders (SOHTEs) for soft tissue expansion (STE). METHODS: A comprehensive search on Pubmed and Google Scholar databases was conducted to identify human studies using SOHTEs for STE; last searched in March 2023. Expansion phase details and expander variables were documented. Complication rates, soft tissue gain, and bone gain reported in each study were also recorded. The inclusion criteria encompassed human studies ranging from evidence levels II-IV (Oxford Centre for Evidence-Based Medicine Levels of Evidence), without specific date limits. For assessing bias in randomized controlled trials (RCTs), a Risk of Bias tool was employed. The synthesised results were presented through tables, sunburst plots, and bar charts. RESULTS: A total of 13 studies were identified, comprising 4 RCTs, 1 cohort study, and 8 case-series. Employment of SOHTEs yielded an overall complication rate of 17% (24/140 sites), with expander perforation accounting for 9.3% (13/140) of the sites. Specific complication rates included dehiscence (1.4%, 2/140 sites), paraesthesia (1.4%, 2/140 sites), and infection (1.4%, 2/140 sites). All randomized controlled trials (RCTs) were categorised at Level II. The remaining investigations primarily consisted of Level IV case-series lacking controls. All studies demonstrated some concerns towards bias. CONCLUSION: STE studies using SOHTEs exhibit a reduction in complications associated with bone augmentation in scenarios of inadequate soft tissue coverage. Preliminary evidence suggests potential benefits even in cases with sufficient soft tissue. Adherence to procedural precautions may reduce the risk of expander perforations, further diminishing complications. Subsequent studies should incorporate individual patient and expander variables in their reports to explore the impact of expansion phases on complication rates, as well as bone and soft tissue augmentation.

Structure of a membrane-bound menaquinol:organohalide oxidoreductase.

Organohalide-respiring bacteria are key organisms for the bioremediation of soils and aquifers contaminated with halogenated organic compounds. The major players in this process are respiratory reductive dehalogenases, corrinoid enzymes that use organohalides as substrates and contribute to energy conservation. Here, we present the structure of a menaquinol:organohalide oxidoreductase obtained by cryo-EM. The membrane-bound protein was isolated from Desulfitobacterium hafniense strain TCE1 as a PceA2B2 complex catalysing the dechlorination of tetrachloroethene. Two catalytic PceA subunits are anchored to the membrane by two small integral membrane PceB subunits. The structure reveals two menaquinone molecules bound at the interface of the two different subunits, which are the starting point of a chain of redox cofactors for electron transfer to the active site. In this work, the structure elucidates how energy is conserved during organohalide respiration in menaquinone-dependent organohalide-respiring bacteria.

Efficacy and safety of using antibiotics to prevent post-operative complications in oral implant treatment: evidence-based review.

AIMS: To identify and critically appraise available evidence on the efficacy and safety of antibiotics in preventing complications following oral implant placement treatment. METHODS: An electronic search was performed using PubMed, Ovid MEDLINE and Cochrane Library databases up to July/21 for the purpose of answering the research question: In[healthy adults treated with dental implants]the use of[different antibiotics before or immediately after treatment]in comparison to[treatment without antibiotics]is safe and effective in terms of[infection, pain, swelling, wound dehiscence, soft tissue healing, early/late implant failure]? Following the Best Evidence Topic methodology, the included studies were categorised based on the Oxford Centre for Evidence-Based Medicine (OCEBM) ratings. The critical appraisal skills programme CASP checklist was used for the methodological analysis. The risk of bias assessment was performed according to the Cochrane Methodology for Systematic Reviews of Interventions. RESULTS: 26 of the 245 initially identified articles met our inclusion criteria for analysis after applying rigorous filters. The included human studies demonstrated significant methodological heterogeneity, precluding meta-analysis. These studies spanned evidence levels II to IV, as per OCEBM 2011 classifications, with the United States contributing the most studies (19.2%, n = 5), all at level III. The United Kingdom and Spain followed with three studies each (11.5% each), two from the UK and one from Spain classified at level II. Most studies had less than 1 year of follow-up (21%). Our analysis included 26 studies, with 38 antibiotic patient groups totalling 7459 patients. Amoxicillin was the predominant antibiotic, with various dosage regimens. Complications were observed in studies across different amoxicillin regimens at a cumulative incidence of 5%. CONCLUSION: The evidence on antibiotics to prevent implant failure presents uncertain and heterogeneous findings. High-risk bias and underpowered studies were prevalent. Future research should prioritise multicentre, double-blinded RCTs with larger samples and longer follow-ups. Structured methodologies, antibiotic stewardship, and adherence to guidelines are needed. Amoxicillin (2 g) was commonly prescribed, but guidelines recommend 3 g, which results in relatively low complications yet there is limited evidence to support it. Clindamycin was favoured for penicillin allergies, but caution is advised due to potential implant failure risk. Consistent use of antiseptic mouthwash was observed. Future research should explore alternatives to antibiotics and antibiotic stewardship. Establishing a well-funded research consortium could yield conclusive results for clinical practice.