Pathogenicity is associated with population structure in a fungal pathogen of humans.

Aspergillus flavus is a clinically and agriculturally important saprotrophic fungus responsible for severe human infections and extensive crop losses. We analyzed genomic data from 250 (95 clinical and 155 environmental) A. flavus isolates from 9 countries, including 70 newly sequenced clinical isolates, to examine population and pan-genome structure and their relationship to pathogenicity. We identified five A. flavus populations, including a new population, D, corresponding to distinct clades in the genome-wide phylogeny. Strikingly, > 75% of clinical isolates were from population D. Accessory genes, including genes within biosynthetic gene clusters, were significantly more common in some populations but rare in others. Population D was enriched for genes associated with zinc ion binding, lipid metabolism, and certain types of hydrolase activity. In contrast to the major human pathogen Aspergillus fumigatus, A. flavus pathogenicity in humans is strongly associated with population structure, making it a great system for investigating how population-specific genes contribute to pathogenicity.

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

High Detection Frequency of Vaccine-Associated Polioviruses and Non-Polio Enteroviruses in the Stools of Asymptomatic Infants from the Free State Province, South Africa.

Enterovirus (EV) infections are widespread and associated with a range of clinical conditions, from encephalitis to meningitis, gastroenteritis, and acute flaccid paralysis. Knowledge about the circulation of EVs in neonatal age and early infancy is scarce, especially in Africa. This study aimed to unveil the frequency and diversity of EVs circulating in apparently healthy newborns from the Free State Province, South Africa (SA). For this purpose, longitudinally collected faecal specimens (May 2021-February 2022) from a cohort of 17 asymptomatic infants were analysed using metagenomic next-generation sequencing. Overall, seven different non-polio EV (NPEV) subtypes belonging to EV-B and EV-C species were identified, while viruses classified under EV-A and EV-D species could not be characterised at the sub-species level. Additionally, under EV-C species, two vaccine-related poliovirus subtypes (PV1 and PV3) were identified. The most prevalent NPEV species was EV-B (16/17, 94.1%), followed by EV-A (3/17, 17.6%), and EV-D (4/17, 23.5%). Within EV-B, the commonly identified NPEV types included echoviruses 6, 13, 15, and 19 (E6, E13, E15, and E19), and coxsackievirus B2 (CVB2), whereas enterovirus C99 (EV-C99) and coxsackievirus A19 (CVA19) were the only two NPEVs identified under EV-C species. Sabin PV1 and PV3 strains were predominantly detected during the first week of birth and 6-8 week time points, respectively, corresponding with the OPV vaccination schedule in South Africa. A total of 11 complete/near-complete genomes were identified from seven NPEV subtypes, and phylogenetic analysis of the three EV-C99 identified revealed that our strains were closely related to other strains from Cameroon and Brazil, suggesting global distribution of these strains. This study provides an insight into the frequency and diversity of EVs circulating in asymptomatic infants from the Free State Province, with the predominance of subtypes from EV-B and EV-C species. This data will be helpful to researchers looking into strategies for the control and treatment of EV infection.

Current Management of Acute and Posttraumatic Critical Bone Defects.

SUMMARY: Limb reconstruction in patients with critical-sized bone defects remains a challenge due to the availability of various technically demanding treatment options and a lack of standardized decision algorithms. Although no consensus exists, it is apparent from the literature that the combination of patient, surgeon, and institutional collaborations is effective in providing the most efficient care pathway for these patients. Success relies on choosing a particular surgical approach that manages infection, soft tissue defects, stability, and alignment. Recent systematic reviews demonstrate high success rates with the following management options: Ilizarov bone transport, Masquelet (induced membrane) technique, cancellous bone grafting, and vascularized bone grafts.

Racial/Ethnic and Gender Diversity of Orthopaedic Journal Editorial Boards.

BACKGROUND: In the current era of evidence-based medicine, scientific publications play a crucial role in guiding patient care. While the lack of diversity among orthopaedic surgeons has been well documented, little is known about the diversity of orthopaedic journal editorial boards. The purpose of this study was to assess the racial/ethnic and gender diversity of U.S. orthopaedic journal editorial boards. METHODS: The editorial boards of 13 orthopaedic journals were examined, including 10 subspecialty and 3 general orthopaedic journals. Race/ethnicity and gender were determined for each editorial board member. The representation observed on orthopaedic journal editorial boards was compared with representation at other phases of the orthopaedic pipeline, as well as within the various subspecialty fields of orthopaedics. Logistic regression and t tests were used to evaluate these comparisons. RESULTS: We identified 876 editorial board members of the 13 journals; 14.0% were Asian, 1.9% were Black, 1.9% were Hispanic, 2.4% were multiracial/other, and 79.7% were White. Racial/ethnic representation was similar across the subspecialty fields of orthopaedics (p > 0.05). The representation of women on orthopaedic editorial boards was 7.9%, with differences in gender diversity observed across subspecialty fields (p < 0.05). Among journals in the subspecialty fields of spine and trauma, female editorial board representation was lower than expected, even after taking into account the representation of women in these subspecialty fields (2.0% versus 9.0% [p = 0.002] and 3.8% versus 10.0% [p = 0.03], respectively). CONCLUSIONS: In this study of 13 subspecialty and general orthopaedic journals, the representation of racial/ethnic minorities and women on editorial boards was similar to their representation in academic orthopaedics. However, these values remain low in comparison with the population of patients treated by orthopaedic surgeons. Given the importance of scientific publications in the current era of evidence-based medicine, orthopaedic journals should continue working to diversify the membership of their editorial boards.

Genomic Analysis of Rwandan G9P[8] Rotavirus Strains Pre- and Post-RotaTeq® Vaccine Reveals Significant Distinct Sub-Clustering in a Post-Vaccination Cohort.

Although the introduction of rotavirus vaccines has substantially contributed to the reduction in rotavirus morbidity and mortality, concerns persist about the re-emergence of variant strains that might alter vaccine effectiveness in the long term. The G9 strains re-emerged in Africa during the mid-1990s and have more recently become predominant in some countries, such as Ghana and Zambia. In Rwanda, during the 2011 to 2015 routine surveillance period, G9P[8] persisted during both the pre- and post-vaccine periods. The pre-vaccination cohort was based on the surveillance period of 2011 to 2012, and the post-vaccination cohort was based on the period of 2013 to 2015, excluding 2014. The RotaTeq® vaccine that was first introduced in Rwanda in 2012 is genotypically heterologous to Viral Protein 7 (VP7) G9. This study elucidated the whole genome of Rwandan G9P[8] rotavirus strains pre- and post-RotaTeq® vaccine introduction. Fecal samples from Rwandan children under the age of five years (pre-vaccine n = 23; post-vaccine n = 7), conventionally genotyped and identified as G9P[8], were included. Whole-genome sequencing was then performed using the Illumina® MiSeq platform. Phylogenetic analysis and pair-wise sequence analysis were performed using MEGA6 software. Distinct clustering of three post-vaccination study strains was observed in all 11 gene segments, compared to the other Rwandan G9P[8] study strains. Specific amino acid differences were identified across the gene segments of these three 2015 post-vaccine strains. Important amino acid differences were identified at position N242S in the VP7 genome segment of the three post-vaccine G9 strains compared to the other G9 strains. This substitution occurs at a neutralization epitope site and may slightly affect protein interaction at that position. These findings indicate that the Rwandan G9P[8] strains revealed a distinct sub-clustering pattern among post-vaccination study strains circulating in Rwanda, with changes at neutralization epitopes, which may play a role in neutralization escape from vaccine candidates. This emphasizes the need for continuous whole-genome surveillance to better understand the evolution and epidemiology of the G9P[8] strains post-vaccination.

Increased Duration and Frequency of Lateral C-Arm Draping Does Not Increase Contamination Rates.

This study sought to quantify the rate of culture-positive drape contamination with varying degrees of drape manipulation for intra-operative fluoroscopic imaging. In this prospective cohort study, 30 patients with operatively closed lower extremity fractures were evaluated. The clip-drape technique was employed to cover the emitter. Swab samples were collected for bacterial growth. A t-test was applied for statistical comparison. Three of 30 cases (10% of operations) showed evidence of contamination. There was no statistically significant difference between duration of drape use or the amount of drape manipulations. None of the 30 patients in this study developed surgical site infection 90-days post-surgery. The clip drape technique for lateral fluoroscopy appears to be effective in maintaining surgical field sterility. Moreover, the number of drape manipulations and length of time the drape was in use was not related to drape contamination. Level of Evidence: Therapeutic Level II. (Journal of Surgical Orthopaedic Advances 32(2):107-110, 2023).

A new and effective method to induce infection of Phyllachora maydis into corn for tar spot studies in controlled environments.

BACKGROUND: Tar spot of corn is a significant and spreading disease in the continental U.S. and Canada caused by the obligate biotrophic fungus Phyllachora maydis. As of 2023, tar spot had been reported in 18 U.S. states and one Canadian Province. The symptoms of tar spot include chlorotic flecking followed by the formation of black stromata where conidia and ascospores are produced. Advancements in research and management for tar spot have been limited by a need for a reliable method to inoculate plants to enable the study of the disease. The goal of this study was to develop a reliable method to induce tar spot in controlled conditions. RESULTS: We induced infection of corn by P. maydis in 100% of inoculated plants with a new inoculation method. This method includes the use of vacuum-collection tools to extract ascospores from field-infected corn leaves, application of spores to leaves, and induction of the disease in the dark at high humidity and moderate temperatures. Infection and disease development were consistently achieved in four independent experiments on different corn hybrids and under different environmental conditions in a greenhouse and growth chamber. Disease induction was impacted by the source and storage conditions of spores, as tar spot was not induced with ascospores from leaves stored dry at 25 ºC for 5 months but was induced using ascospores from infected leaves stored at -20 ºC for 5 months. The time from inoculation to stromata formation was 10 to 12 days and ascospores were present 19 days after inoculation throughout our experiments. In addition to providing techniques that enable in-vitro experimentation, our research also provides fundamental insights into the conditions that favor tar spot epidemics. CONCLUSIONS: We developed a method to reliably inoculate corn with P. maydis. The method was validated by multiple independent experiments in which infection was induced in 100% of the plants, demonstrating its consistency in controlled conditions. This new method facilitates research on tar spot and provides opportunities to study the biology of P. maydis, the epidemiology of tar spot, and for identifying host resistance.

Mining for a new class of fungal natural products: the evolution, diversity, and distribution of isocyanide synthase biosynthetic gene clusters.

The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We amalgamated a pipeline of tools to predict BGCs based on shared promoter motifs and located 3800 ICS BGCs in 3300 genomes, making ICS BGCs the fifth largest class of specialized metabolites compared to canonical classes found by antiSMASH. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1/2 gene cluster family (GCF), which was prior only studied in yeast, is present in ∼30% of all Ascomycetes. The dit variety ICS exhibits greater similarity to bacterial ICS than other fungal ICS, suggesting a potential convergence of the ICS backbone domain. The evolutionary origins of the dit GCF in Ascomycota are ancient and these genes are diversifying in some lineages. Our results create a roadmap for future research into ICS BGCs. We developed a website (https://isocyanides.fungi.wisc.edu/) that facilitates the exploration and downloading of all identified fungal ICS BGCs and GCFs.

Coupling proteins, with deadly consequences.

Phage protein E shuts down cell wall biosynthesis to kill bacteria.

Aneuploidy Formation in the Filamentous Fungus Aspergillus flavus in Response to Azole Stress.

Aspergillus flavus is a mycotoxigenic fungus that contaminates many important agricultural crops with aflatoxin B1, the most toxic and carcinogenic natural compound. This fungus is also the second leading cause of human invasive aspergillosis, after Aspergillus fumigatus, a disease that is particularly prevalent in immunocompromised individuals. Azole drugs are considered the most effective compounds in controlling Aspergillus infections both in clinical and agricultural settings. Emergence of azole resistance in Aspergillus spp. is typically associated with point mutations in cyp51 orthologs that encode lanosterol 14α-demethylase, a component of the ergosterol biosynthesis pathway that is also the target of azoles. We hypothesized that alternative molecular mechanisms are also responsible for acquisition of azole resistance in filamentous fungi. We found that an aflatoxin-producing A. flavus strain adapted to voriconazole exposure at levels above the MIC through whole or segmental aneuploidy of specific chromosomes. We confirm a complete duplication of chromosome 8 in two sequentially isolated clones and a segmental duplication of chromosome 3 in another clone, emphasizing the potential diversity of aneuploidy-mediated resistance mechanisms. The plasticity of aneuploidy-mediated resistance was evidenced by the ability of voriconazole-resistant clones to revert to their original level of azole susceptibility following repeated transfers on drug-free media. This study provides new insights into mechanisms of azole resistance in a filamentous fungus. IMPORTANCE Fungal pathogens cause human disease and threaten global food security by contaminating crops with toxins (mycotoxins). Aspergillus flavus is an opportunistic mycotoxigenic fungus that causes invasive and noninvasive aspergillosis, diseases with high rates of mortality in immunocompromised individuals. Additionally, this fungus contaminates most major crops with the notorious carcinogen, aflatoxin. Voriconazole is the drug of choice to treat infections caused by Aspergillus spp. Although azole resistance mechanisms have been well characterized in clinical isolates of Aspergillus fumigatus, the molecular basis of azole resistance in A. flavus remains unclear. Whole-genome sequencing of eight voriconazole-resistant isolates revealed that, among other factors, A. flavus adapts to high concentrations of voriconazole by duplication of specific chromosomes (i.e., aneuploidy). Our discovery of aneuploidy-mediated resistance in a filamentous fungus represents a paradigm shift, as this type of resistance was previously thought to occur only in yeasts. This observation provides the first experimental evidence of aneuploidy-mediated azole resistance in the filamentous fungus A. flavus.

Orthopaedic Advances: Use of Three-Dimensional Metallic Implants for Reconstruction of Critical Bone Defects After Trauma.

Multiple successful strategies exist for the management of critical-sized bone defects. Depending on the location and etiology of an osseous defect, there are nuances that must be considered by the treating surgeon. The induced membrane technique and various modifications of the Ilizarov method (bone transport by distraction osteogenesis) have been the most common methods for biologic reconstruction. Despite the versatility and high union rates reported, they may not be practical for every patient. The rapid expansion of three-dimensional printing of medical devices has led to an increase in their use within orthopaedic surgery, specifically in the definitive treatment of critical bone defects. This article proposes indications and contraindications for implementation of this technology and reviews the available clinical evidence on the use of custom nonresorbable implants for the treatment of traumatic bone loss. Clinical cases are presented to illustrate the scenarios in which this approach is viable.

Racial and Sex Disparities in Resident Attrition in Orthopaedic Surgery.

Studies have suggested that female individuals and individuals from backgrounds under-represented in medicine (URiM) are at increased risk of attrition during residency. This likely exacerbates the lack of diversity in our field. The aims of this study were to (1) characterize demographic composition in orthopaedic residency from 2001 to 2018 and (2) determine the race/ethnicity and identify any disparities. Methods: Demographic and attrition data from 2001 to 2018 were obtained from the Association of American Medical Colleges. Attrition data comprised the following categories: withdrawals, dismissals, and transfers to another specialty. Analysis compared demographic composition and determined attrition rates with subgroup analysis by race/ethnicity and sex. Results: From 2001 to 2018, female orthopaedic residents increased from 8.77% to 15.54% and URiM residents from 9.49% to 11.32%. The overall and unintended attrition rates in orthopaedic surgery were 3.20% and 1.15%, respectively. Among female residents, the overall and unintended attrition rates were 5.96% and 2.09% compared with 2.79% and 1.01%, respectively, in male residents. URiM residents had overall and unintended attrition rates of 6.16% and 3.11% compared with 2.71% and 0.83%, respectively, for their White counterparts. Black/African American residents had an attrition rate of nearly 10%. Female residents averaged 12.9% of all residents but 24% of those leaving orthopaedics. URiM residents were 10.14% of all residents but 19.51% of those experiencing attrition. In logistic regression models, female residents had a relative risk (RR) of 2.20 (p < 0.001) for experiencing all-cause attrition and 2.09 (p < 0.001) for unintended attrition compared with male residents. Compared with their White male counterparts, URiM residents had a RR for overall and unintended attrition of 2.36 and 3.84 (p < 0.001), respectively; Black/African American residents had a RR for the same of 3.80 and 7.20 (p < 0.001), respectively. Conclusion: Although female resident percentage has increased, orthopaedics continues to train fewer female surgeons than all other fields. Female and URiM residents in orthopaedic surgery are disproportionately affected by attrition. While recruitment has been the primary focus of diversity, equity, and inclusion efforts, this study suggests that resident retention through appropriately supporting residents during training is equally critical.

How do surgeons decide when to treat proximal humerus fractures with operative versus nonoperative management?

PURPOSE: The objective of this study was to determine the underlying factors that drive the decision for surgeons to pursue operative versus nonoperative management for proximal humerus fractures (PHF) and if fellowship training had an impact on these decisions. METHODS: An electronic survey was distributed to members of the Orthopaedic Trauma Association and the American Shoulder and Elbow Surgeons Society to assess differences in patient selection for operative versus nonoperative management of PHF. Descriptive statistics were reported for all respondents. RESULTS: A total of 250 fellowship trained Orthopaedic Surgeons responded to the online survey. A greater proportion of trauma surgeons preferred nonoperative management for displaced PHF fractures in patients over the age of 70. Operative management was preferred for older patients with fracture dislocations (98%), limited humeral head bone subchondral bone (78%), and intraarticular head split (79%). Similar proportions of trauma surgeons and shoulder surgeons cited that acquiring a CT was crucial to distinguish between operative and nonoperative management. CONCLUSION: We found that surgeons base their decisions on when to operate primarily on patient's comorbidities, age, and the amount of fracture displacement when treating younger patients. Further, we found a greater proportion of trauma surgeons elected to proceed with nonoperative management in patients older than the age of 70 years old as compared to shoulder surgeons.

LaeA-Regulated Fungal Traits Mediate Bacterial Community Assembly.

Potent antimicrobial metabolites are produced by filamentous fungi in pure culture, but their ecological functions in nature are often unknown. Using an antibacterial Penicillium isolate and a cheese rind microbial community, we demonstrate that a fungal specialized metabolite can regulate the diversity of bacterial communities. Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA deletion strain had significantly higher bacterial abundances than the wild-type strain. RNA-sequencing and metabolite profiling demonstrated a striking reduction in the expression and production of the natural product pseurotin in the laeA deletion strain. Inactivation of a core gene in the pseurotin biosynthetic cluster restored bacterial community composition, confirming the role of pseurotins in mediating bacterial community assembly. Our discovery demonstrates how global regulators of fungal transcription can control the assembly of bacterial communities and highlights an ecological role for a widespread class of fungal specialized metabolites. IMPORTANCE Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Our study highlights how specialized metabolites secreted by a Penicillium species can mediate cheese rind development via differential inhibition of bacterial community members. Because LaeA regulates specialized metabolites and other ecologically relevant traits in a wide range of filamentous fungi, this global regulator may have similar impacts in other fungus-dominated microbiomes.

Lipase-catalysed changes in essential oils revealed by comprehensive two-dimensional gas chromatography.

Candida antarctica lipase A (CALA) was applied for the chemo-selective enzymatic transesterification of terpene and phenyl alcohols in 35 different essential oil samples. Comprehensive two-dimensional gas chromatography with mass spectrometry (GC×GC‒MS) analysis enabled the separation and tentative identification of a cohort of 125 compounds, allowing the instant visualisation of the reaction process changes, amid the complex chemical background of the samples. The results indicate that 42 out of 79 alcohols so-identified were fully or partially esterified within 48 h of reaction, with primary alcohols being the substrates of preference of the enzyme (90-100% conversion), followed by secondary alcohols (mostly ~ 80-100% conversion). No significant conversion of tertiary alcohols and phenols was observed using the tested conditions. Overall, the enzyme's performance was consistent for primary alcohol substrates identified in multiple samples of different compositions. The observed selectivity, efficiency, robustness, scalability (enzyme/substrate working concentration ratio > 1:160), potential reusability, mild reaction conditions, and other factors make this process a greener and more sustainable alternative for industry applications, particularly for the manufacture of novel flavours and fragrances.

The Evolution of Post-Vaccine G8P[4] Group a Rotavirus Strains in Rwanda; Notable Variance at the Neutralization Epitope Sites.

Africa has a high level of genetic diversity of rotavirus strains, which is suggested to be a possible reason contributing to the suboptimal effectiveness of rotavirus vaccines in this region. One strain that contributes to this rotavirus diversity in Africa is the G8P[4]. This study aimed to elucidate the entire genome and evolution of Rwandan G8P[4] strains. Illumina sequencing was performed for twenty-one Rwandan G8P[4] rotavirus strains. Twenty of the Rwandan G8P[4] strains had a pure DS-1-like genotype constellation, and one strain had a reassortant genotype constellation. Notable radical amino acid differences were observed at the neutralization sites when compared with cognate regions in vaccine strains potentially playing a role in neutralization escape. Phylogenetic analysis revealed that the closest relationship was with East African human group A rotavirus (RVA) strains for five of the genome segments. Two genome sequences of the NSP4 genome segment were closely related to bovine members of the DS-1-like family. Fourteen VP1 and eleven VP3 sequences had the closest relationships with the RotaTeq™ vaccine WC3 bovine genes. These findings suggest that the evolution of VP1 and VP3 might have resulted from reassortment events with RotaTeq™ vaccine WC3 bovine genes. The close phylogenetic relationship with East African G8P[4] strains from Kenya and Uganda suggests co-circulation in these countries. These findings highlight the need for continued whole-genomic surveillance to elucidate the evolution of G8P[4] strains, especially after the introduction of rotavirus vaccination.

Pangenomics of the death cap mushroom Amanita phalloides, and of Agaricales, reveals dynamic evolution of toxin genes in an invasive range.

The poisonous European mushroom Amanita phalloides (the "death cap") is invading California. Whether the death caps' toxic secondary metabolites are evolving as it invades is unknown. We developed a bioinformatic pipeline to identify the MSDIN genes underpinning toxicity and probed 88 death cap genomes from an invasive Californian population and from the European range, discovering a previously unsuspected diversity of MSDINs made up of both core and accessory elements. Each death cap individual possesses a unique suite of MSDINs, and toxin genes are significantly differentiated between Californian and European samples. MSDIN genes are maintained by strong natural selection, and chemical profiling confirms MSDIN genes are expressed and result in distinct phenotypes; our chemical profiling also identified a new MSDIN peptide. Toxin genes are physically clustered within genomes. We contextualize our discoveries by probing for MSDINs in genomes from across the order Agaricales, revealing MSDIN diversity originated in independent gene family expansions among genera. We also report the discovery of an MSDIN in an Amanita outside the "lethal Amanitas" clade. Finally, the identification of an MSDIN gene and its associated processing gene (POPB) in Clavaria fumosa suggest the origin of MSDINs is older than previously suspected. The dynamic evolution of MSDINs underscores their potential to mediate ecological interactions, implicating MSDINs in the ongoing invasion. Our data change the understanding of the evolutionary history of poisonous mushrooms, emphasizing striking parallels to convergently evolved animal toxins. Our pipeline provides a roadmap for exploring secondary metabolites in other basidiomycetes and will enable drug prospecting.

Comparative yields of antimicrobial peptides released from human and cow milk proteins under infant digestion conditions predicted by in silico methodology.

Mammalian milk proteins are known to encrypt antimicrobial peptides (AMPs) which can be passively released and exert bioactivity in the gastrointestinal and cardiovascular systems pre- or post-absorption, respectively. However, the contribution of 'passive' food-derived AMPs to the pool of endogenous and microbial AMPs has not been differentiated in previous research. Insight into the consequences of protein digestion and peptide bioactivity can be gained using in silico tools. The aim of this investigation was to use in silico methods to characterise the yields of AMPs released from major proteins in human and cow milk under infant digestion conditions, as relevant to early nutrition. The profiles of major proteins in human and cow milk from UniProtKB/Swiss-Prot, were subjected to in silico digestion by ExPASy-PeptideCutter, and the AMP activity of resulting peptides (≥4 amino acids, AAs) evaluated with the CAMPR3-RF predictive tool. The mass yields and counts of absorbing (≤10 AAs) and non-absorbing (>10 AAs) AMPs, as found in human, cow and 'humanised' ratios of cow milk proteins, were quantified. The results indicated that major whey proteins from both human and cow milks displayed a higher degree of hydrolysis than caseins, consistent with their known 'fast' digestion properties. Larger albumin and lactoferrin proteins generated relatively more and/or longer peptides. Yields of AMPs from cow milk were higher than from human milk, even after standardising the ratio of whey to casein and total protein concentration, as practiced in formulations manufactured for human newborn babies. Whereas alpha-lactalbumin (2.65 g L-1) and lactoferrin (1.75 g L-1) provided the major yields of AMPs in human milk whey proteins; beta-lactoglobulin, which is unique to cow milk, released the highest yield of AMPs in cow milk (3.25 g L-1 or 19.9% w/w of total whey protein), which may represent an important and overlooked biological function of this protein in cow milk.

Mining for a New Class of Fungal Natural Products: The Evolution, Diversity, and Distribution of Isocyanide Synthase Biosynthetic Gene Clusters.

The products of non-canonical isocyanide synthase (ICS) biosynthetic gene clusters (BGCs) have notable bioactivities that mediate pathogenesis, microbial competition, and metal-homeostasis through metal-associated chemistry. We sought to enable research into this class of compounds by characterizing the biosynthetic potential and evolutionary history of these BGCs across the Fungal Kingdom. We developed the first genome-mining pipeline to identify ICS BGCs, locating 3,800 ICS BGCs in 3,300 genomes. Genes in these clusters share promoter motifs and are maintained in contiguous groupings by natural selection. ICS BGCs are not evenly distributed across fungi, with evidence of gene-family expansions in several Ascomycete families. We show that the ICS dit1 / 2 gene cluster family (GCF), which was thought to only exist in yeast, is present in ∻30% of all Ascomycetes, including many filamentous fungi. The evolutionary history of the dit GCF is marked by deep divergences and phylogenetic incompatibilities that raise questions about convergent evolution and suggest selection or horizontal gene transfers have shaped the evolution of this cluster in some yeast and dimorphic fungi. Our results create a roadmap for future research into ICS BGCs. We developed a website ( www.isocyanides.fungi.wisc.edu ) that facilitates the exploration, filtering, and downloading of all identified fungal ICS BGCs and GCFs.