A cryptic site in class 5 epitope of SARS-CoV-2 RBD maintains highly conservation across natural isolates.

The emergence of SARS-CoV-2 variants raises concerns about the efficacy of existing COVID-19 vaccines and therapeutics. Previously, we identified a conserved cryptic class 5 epitope of SARS-CoV-2 receptor binding domain (RBD) by two cross-neutralizing antibodies 7D6 and 6D6. Intriguingly, this site remains resistant to substantial mutations occurred in ever-changing SARS-CoV-2 subvariants. As compared to class 3 antibody S309, 6D6 maintains broad and consistent neutralizing activities against SARS-CoV-2 variants. Furthermore, 6D6 effectively protected hamster from the virulent Beta strain. Sequence alignment of approximately 6 million documented SARS-CoV-2 isolates revealed that 6D6 epitope maintains an exceptionally high conservation rate (99.92%). Structural analysis demonstrated that all 33 mutations accumulated in XBB.1.5 since the original strain do not perturb the binding 6D6 to RBD, in line with the sequence analysis throughout the antigenicity evolution of SARS-CoV-2. These findings suggest the potential of this epitope serving as a critical determinant for vaccines and therapeutic design.

Comprehensive Overview of Broadly Neutralizing Antibodies against SARS-CoV-2 Variants.

Currently, SARS-CoV-2 has evolved into various variants, including the numerous highly mutated Omicron sub-lineages, significantly increasing immune evasion ability. The development raises concerns about the possibly diminished effectiveness of available vaccines and antibody-based therapeutics. Here, we describe those representative categories of broadly neutralizing antibodies (bnAbs) that retain prominent effectiveness against emerging variants including Omicron sub-lineages. The molecular characteristics, epitope conservation, and resistance mechanisms of these antibodies are further detailed, aiming to offer suggestion or direction for the development of therapeutic antibodies, and facilitate the design of vaccines with broad-spectrum potential.

Novel bidentate N-coordinated alkylaluminum complexes: synthesis, characterization, and efficient catalysis for hydrophosphonylation.

Five new alkylaluminum complexes with different pyridinyl-substituted imines or cyclohexyl-substituted imines were synthesized and characterized successfully. The aluminum complex [FlCHNCH(CH3)Py]AlMe2(Py = 2-pyridyl) (1) was obtained by reacting 9-[2-pyridyl-CH(CH3)-NCH]Fl (Fl = fluorenyl) (L1) and equimolar AlMe3. The reactions of 9-(2-pyridyl-NCH)Fl (L2) and 9-[2-N(CH3)2-cyclohexyl-NCH]Fl (L3) with equimolar AlMe3 or AlEt3 afforded other alkylaluminum complexes [FlCHNPy]AlMe2(Py = 2-pyridyl) (2), [FlCHNPy]AlEt2 (Py = 2-pyridyl) (3), [FlCHNCyN(CH3)2]AlMe2 (Cy = 2-cyclohexyl) (4) and [FlCHNCyN(CH3)2]AlEt2 (Cy = 2-cyclohexyl) (5). All these complexes (1-5) were characterized using NMR spectroscopy, elemental analysis, and X-ray crystal structure analysis. The catalytic properties of these new alkylaluminum complexes for the hydrophosphonylation of aldimines were examined. Complex 5 showed the best catalytic performance under mild reaction conditions with a low catalyst loading (1 mol%), and 20 different substituents of aldimines were isolated with more than 90% yields.

HHCDB: a database of human heterochromatin regions.

Heterochromatin plays essential roles in eukaryotic genomes, such as regulating genes, maintaining genome integrity and silencing repetitive DNA elements. Identifying genome-wide heterochromatin regions is crucial for studying transcriptional regulation. We propose the Human Heterochromatin Chromatin Database (HHCDB) for archiving heterochromatin regions defined by specific or combined histone modifications (H3K27me3, H3K9me2, H3K9me3) according to a unified pipeline. 42 839 743 heterochromatin regions were identified from 578 samples derived from 241 cell-types/cell lines and 92 tissue types. Genomic information is provided in HHCDB, including chromatin location, gene structure, transcripts, distance from transcription start site, neighboring genes, CpG islands, transposable elements, 3D genomic structure and functional annotations. Furthermore, transcriptome data from 73 single cells were analyzed and integrated to explore cell type-specific heterochromatin-related genes. HHCDB affords rich visualization through the UCSC Genome Browser and our self-developed tools. We have also developed a specialized online analysis platform to mine differential heterochromatin regions in cancers. We performed several analyses to explore the function of cancer-specific heterochromatin-related genes, including clinical feature analysis, immune cell infiltration analysis and the construction of drug-target networks. HHCDB is a valuable resource for studying epigenetic regulation, 3D genomics and heterochromatin regulation in development and disease. HHCDB is freely accessible at http://hhcdb.edbc.org/.

Efficacy and safety of third-line or later-line targeted treatment for patients with metastatic colorectal cancer: a meta-analysis.

Targeted therapy has been standardized in front-line therapies for metastatic colorectal cancer (mCRC), while explicit recommendations for third- or later-line are still lacking. This study evaluated the efficacy and safety of combining targeted therapy with chemotherapy in the third- or later-line treatment for mCRC via meta-analysis, providing evidence-based guidance for clinical or research practice. Comprehensive retrieval of related studies was conducted according to the PRISMA guideline. Studies were stratified with patient characteristics and pharmacological classification of the drugs. For the data available for quantitative analysis, pooled overall response rate, disease control rate, hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS), and adverse events rate with respective 95% confidence intervals (CIs) were calculated. A total of 22 studies (1,866 patients) were included in this meta-analysis. Data from 17 studies (1,769 patients) involving targets of epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) were extracted for meta-analyses. The overall response rates for monotherapy and combined therapy were 4% (95% CI: 3%, 5%) and 20% (95% CI: 11%, 29%). The pooled HRs (combined therapy vs. mono) for OS and PFS were 0.72 (95% CI: 0.53, 0.99) and 0.34 (95% CI: 0.26, 0.45). Another five studies were included in narrative depiction, involving targets of BRAF, HER-2, ROS1, and NTRK. The findings of this meta-analysis indicate that VEGF and EGFR inhibitors manifest promising clinical response rates and prolonged survival in the treatment of mCRC with acceptable adverse events.

Molecular engineering tools for the development of vaccines against infectious diseases: current status and future directions.

INTRODUCTION: The escalating global changes have fostered conditions for the expansion and transmission of diverse biological factors, leading to the rise of emerging and reemerging infectious diseases. Complex viral infections, such as COVID-19, influenza, HIV, and Ebola, continue to surface, necessitating the development of effective vaccine technologies. AREAS COVERED: This review article highlights recent advancements in molecular biology, virology, and genomics that have propelled the design and development of innovative molecular tools. These tools have promoted new vaccine research platforms and directly improved vaccine efficacy. The review summarizes the cutting-edge molecular engineering tools used in creating novel vaccines and explores the rapidly expanding molecular tools landscape and potential directions for future vaccine development. EXPERT OPINION: The strategic application of advanced molecular engineering tools can address conventional vaccine limitations, enhance the overall efficacy of vaccine products, promote diversification in vaccine platforms, and form the foundation for future vaccine development. Prioritizing safety considerations of these novel molecular tools during vaccine development is crucial.

Design of Multicomponent Peptide Fibrils with Ordered and Programmable Compositional Patterns.

Advanced applications of biomacromolecular assemblies require a stringent degree of control over molecular arrangement, which is a challenge to current synthetic methods. Here we used a neighbor-controlled patterning strategy to build multicomponent peptide fibrils with an unprecedented capacity to manipulate local composition and peptide positions. Eight peptides were designed to have regulable nearest neighbors upon co-assembly, which, by simulation, afforded 412 different patterns within fibrils, with varied compositions and/or peptide positions. The fibrils with six prescribed patterns were experimentally constructed with high accuracy. The controlled patterning also applies to functionalities appended to the peptides, as exemplified by arranging carbohydrate ligands at nanoscale precision for protein recognition. This study offers a route to molecular editing of inner structures of peptide assemblies, prefiguring the uniqueness and richness of patterning-based material design.

Truncated glycoprotein E of varicella-zoster virus is an ideal immunogen for Escherichia coli-based vaccine design.

Varicella-zoster virus (VZV) is a highly infectious agent responsible for both varicella and herpes zoster disease. Despite high efficacy, there remain safety and accessibility concerns with the licensed vaccines. Here, we sought to produce a VZV gE immunogen using an E. coli expression system. We found that the soluble expression and yield of gE protein could be enhanced via C-terminal truncations to the protein, thereby facilitating a robust and scalable purification process for the purpose of vaccine manufacturing. The lead truncated gE (aa 31-358), hereafter referred to as tgE, was a homogenous monomer in solution and showed excellent antigenicity. Finally, we assessed and compared the immunogenicity of tgE with commercial vOka LAV and Shingrix vaccine. We found that aluminum-adjuvanted tgE was immunogenic as compared with vOka LAV. When adjuvanted with AS01B, a two-dose immunization of tgE showed comparable or better potency in antibody responses and cell-mediated immunity with those of the Shingrix vaccine at the same dosage, especially in terms of the proportion of IFN-γ-expressing CD4+ T cells. In conclusion, this method of E. coli-mediate tgE expression offers a cost-effective and scalable strategy to generate an ideal VZV gE immunogen for the development of both varicella and zoster vaccines.

Genetic engineering of baculovirus-insect cell system to improve protein production.

The Baculovirus Expression Vector System (BEVS), a mature foreign protein expression platform, has been available for decades, and has been effectively used in vaccine production, gene therapy, and a host of other applications. To date, eleven BEVS-derived products have been approved for use, including four human vaccines [Cervarix against cervical cancer caused by human papillomavirus (HPV), Flublok and Flublok Quadrivalent against seasonal influenza, Nuvaxovid/Covovax against COVID-19], two human therapeutics [Provenge against prostate cancer and Glybera against hereditary lipoprotein lipase deficiency (LPLD)] and five veterinary vaccines (Porcilis Pesti, BAYOVAC CSF E2, Circumvent PCV, Ingelvac CircoFLEX and Porcilis PCV). The BEVS has many advantages, including high safety, ease of operation and adaptable for serum-free culture. It also produces properly folded proteins with correct post-translational modifications, and can accommodate multi-gene- or large gene insertions. However, there remain some challenges with this system, including unstable expression and reduced levels of protein glycosylation. As the demand for biotechnology increases, there has been a concomitant effort into optimizing yield, stability and protein glycosylation through genetic engineering and the manipulation of baculovirus vector and host cells. In this review, we summarize the strategies and technological advances of BEVS in recent years and explore how this will be used to inform the further development and application of this system.

Identification of a cross-neutralizing antibody that targets the receptor binding site of H1N1 and H5N1 influenza viruses.

Influenza A viruses pose a significant threat globally each year, underscoring the need for a vaccine- or antiviral-based broad-protection strategy. Here, we describe a chimeric monoclonal antibody, C12H5, that offers neutralization against seasonal and pandemic H1N1 viruses, and cross-protection against some H5N1 viruses. Notably, C12H5 mAb offers broad neutralizing activity against H1N1 and H5N1 viruses by controlling virus entry and egress, and offers protection against H1N1 and H5N1 viral challenge in vivo. Through structural analyses, we show that C12H5 engages hemagglutinin (HA), the major surface glycoprotein on influenza, at a distinct epitope overlapping the receptor binding site and covering the 140-loop. We identified eight highly conserved (~90%) residues that are essential for broad H1N1 recognition, with evidence of tolerance for Asp or Glu at position 190; this site is a molecular determinant for human or avian host-specific recognition and this tolerance endows C12H5 with cross-neutralization potential. Our results could benefit the development of antiviral drugs and the design of broad-protection influenza vaccines.

Baculovirus Display of Varicella-Zoster Virus Glycoprotein E Induces Robust Humoral and Cellular Immune Responses in Mice.

Varicella-zoster virus (VZV) is the causative agent of varicella and herpes zoster (HZ) and can pose a significant challenge to human health globally. The initial VZV infection-more common in children-causes a self-limiting chicken pox. However, in later life, the latent VZV can become reactivated in these patients, causing HZ and postherpetic neuralgia (PHN), a serious and painful complication. VZV glycoprotein E (gE) has been developed into a licensed subunit vaccine against HZ (Shingrix). However, its efficacy relies on the concomitant delivery of a robust adjuvant (AS01B). Here, we sought to create a new immunogen for vaccine design by displaying the VZV-gE on the baculovirus surface (Bac-gE). Correct localization and display of gE on the engineered baculovirus was verified by flow cytometry and immune electron microscopy. We show that Bac-gE provides excellent antigenicity against VZV and induces not only stronger gE-specific CD4+ and CD8+ T cell responses but also higher levels of VZV-specific neutralizing antibodies as compared with other vaccine strategies in mice. Collectively, we show that the baculovirus display of VZV-gE confers ideal humoral and cellular immune responses required for HZ vaccine development, paving the way for a baculovirus-based vaccine design.

DNA Methylation-Specific Analysis of G Protein-Coupled Receptor-Related Genes in Pan-Cancer.

Tumor heterogeneity presents challenges for personalized diagnosis and treatment of cancer. The identification method of cancer-specific biomarkers has important applications for the diagnosis and treatment of cancer types. In this study, we analyzed the pan-cancer DNA methylation data from TCGA and GEO, and proposed a computational method to quantify the degree of specificity based on the level of DNA methylation of G protein-coupled receptor-related genes (GPCRs-related genes) and to identify specific GPCRs DNA methylation biomarkers (GRSDMs) in pan-cancer. Then, a ridge regression-based method was used to discover potential drugs through predicting the drug sensitivities of cancer samples. Finally, we predicted and verified 8 GRSDMs in adrenocortical carcinoma (ACC), rectum adenocarcinoma (READ), uveal Melanoma (UVM), thyroid carcinoma (THCA), and predicted 4 GRSDMs (F2RL3, DGKB, GRK5, PIK3R6) which were sensitive to 12 potential drugs. Our research provided a novel approach for the personalized diagnosis of cancer and informed individualized treatment decisions.

A Bacterially Expressed SARS-CoV-2 Receptor Binding Domain Fused With Cross-Reacting Material 197 A-Domain Elicits High Level of Neutralizing Antibodies in Mice.

The Coronavirus disease 2019 (COVID-19) pandemic presents an unprecedented public health crisis worldwide. Although several vaccines are available, the global supply of vaccines, particularly within developing countries, is inadequate, and this necessitates a need for the development of less expensive, accessible vaccine options. To this end, here, we used the Escherichia coli expression system to produce a recombinant fusion protein comprising the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; residues 319-541) and the fragment A domain of Cross-Reacting Material 197 (CRM197); hereafter, CRMA-RBD. We show that this CRMA-RBD fusion protein has excellent physicochemical properties and strong reactivity with COVID-19 convalescent sera and representative neutralizing antibodies (nAbs). Furthermore, compared with the use of a traditional aluminum adjuvant, we find that combining the CRMA-RBD protein with a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH-002C-Ac) leads to stronger humoral immune responses in mice, with 4-log neutralizing antibody titers. Overall, our study highlights the value of this E. coli-expressed fusion protein as an alternative vaccine candidate strategy against COVID-19.

Research Note: Study on the antibacterial activity of Chinese herbal medicine against Aspergillus flavus and Aspergillus fumigatus of duck origin in laying hens.

Aspergillus flavus and Aspergillus fumigatus were derived and identified from the ducks infected with fungi. In order to investigate the effectiveness of Chinese herbal medicines against Aspergillus flavus and Aspergillus fumigatus, in vitro antibacterial test and animal infection control test were conducted to study the antibacterial activity of the Chinese medicine mixture which was compatible with Acorus gramineus, Phellodendron chinensis, and Cassia obtusifolia. According to the results, the liver of chickens infected with Aspergillus flavus and Aspergillus fumigatus displayed granulomatous lesions, indicating that the isolation of pathogen from the lungs of sick ducks is also pathogenic to chickens. As suggested by the results of in vitro drug sensitivity test, the mixture 1 MIC80 was the minimum, the MIC80 of Aspergillus flavus was 16 µg/µL, and the MIC80 of Aspergillus fumigatus was 4 µg/µL. In a petri dish of the same concentration, the colony diameter of Aspergillus flavus and Aspergillus fumigatus in Mixture 1 was the minimum. Besides, Aspergillus flavus colonies grew when the concentration was 64 µg/µL, and Aspergillus fumigatus colonies grew when the concentration was 4 µg/µL, which suggests the more significant inhibitory effect of Mixture 1 on Aspergillus flavus and Aspergillus fumigatus. According to the results of animal experiments, there was a significantly lower activity level of Glutamic oxaloacetic transaminase (GOT) and Glutamate pyruvic transaminase (GPT) in the protection group and the treatment group than in the bacterial infection group. As indicated by the blood smear results, there were more neutrophils in the infected group than in the prevention group and the treatment group. Thus, it can be seen from that the Mixture 1 produced preventive and therapeutic effects on the chickens infected with Aspergillus flavus and Aspergillus fumigatus.

The functions of Patchouli and Elsholtzia in the repair of hen follicular granular cells after heat stress.

The objective of this experimental study was to examine the effects of the Chinese herbal medicines Patchouli and Elsholtzia on the follicular granulosa cells of hens undergoing heat stress conditions. In the current investigation, hen follicular granulosa cells were isolated from the prehierarchical follicles of layer hens and then cultured in-vitro. The cells were randomly divided into the 6 groups. Following the completion of this study's experiments using different heat stress and medicinal treatments, the cell activities of each group were measured using an MTT method. The levels of the heat shock protein 70 (HSP70) were detected using ELISA. The expressions of the steroidogenic acute regulatory protein (StAR) mRNA; cytochrome P450 family 11, subfamily A, member 1 (CYP11A1) mRNA; proliferating cell nuclear antigen (PCNA) mRNA; and the follicle stimulating hormone receptor (FSHR) were detected using the real-time quantitative polymerase chain reactions. The concentration levels of estrogen and progesterone in the cell supernatant of each group were measured using ELISA. The results showed that cell activity had significantly decreased following the heat stress treatments at 43℃, 44℃, and 45℃ (P < 0.01), respectively. Meanwhile, cell activities observed in Patchouli and Elsholtzia were found to be much better than those of heat stress group (P < 0.05). In addition, the expression levels of HSP70 in the follicular granulosa cells of Patchouli and Elsholtzia groups were lower than those of heat stress group. Patchouli and Elsholtzia can maintain expressions of the receptor at 43℃. This study determined that the estrogen and progesterone in the supernatant fluid of Patchouli and Elsholtzia were higher than those observed in heat stress. Therefore, the results obtained in this study indicated that the Patchouli and Elsholtzia treatments administered prior the heat stress experiments had successfully protected the follicular granulosa cells from heat damages while maintaining the normal secretory functions of the granulosa cells.

Cross-neutralizing antibodies bind a SARS-CoV-2 cryptic site and resist circulating variants.

The emergence of numerous variants of SARS-CoV-2, the causative agent of COVID-19, has presented new challenges to the global efforts to control the COVID-19 pandemic. Here, we obtain two cross-neutralizing antibodies (7D6 and 6D6) that target Sarbecoviruses' receptor-binding domain (RBD) with sub-picomolar affinities and potently neutralize authentic SARS-CoV-2. Crystal structures show that both antibodies bind a cryptic site different from that recognized by existing antibodies and highly conserved across Sarbecovirus isolates. Binding of these two antibodies to the RBD clashes with the adjacent N-terminal domain and disrupts the viral spike. Both antibodies confer good resistance to mutations in the currently circulating SARS-CoV-2 variants. Thus, our results have direct relevance to public health as options for passive antibody therapeutics and even active prophylactics. They can also inform the design of pan-sarbecovirus vaccines.

Accurate Prediction of Prognosis by Integrating Clinical and Molecular Characteristics in Colon Cancer.

Various factors affect the prognosis of patients with colon cancer. Complicated factors are found to be conducive to accurate assessment of prognosis. In this study, we developed a series of prognostic prediction models for survival time of colon cancer patients after surgery. Analysis of nine clinical characteristics showed that the most important factor was the positive lymph node ratio (LNR). High LNR was the most important clinical factor affecting 1- and 3-year survival; M0&age < 70 was the most important feature for 5 years. The performance of the model was improved through the integration of clinical characteristics and four types of molecule features (mRNA, lncRNA, miRNA, DNA methylation). The model provides guidance for clinical practice. According to the high-risk molecular features combined with age ≥ 70&T3, poorly differentiated or undifferentiated, M0&well differentiated, M0&T2, LNR high, T4&poorly differentiated, or undifferentiated, the survival time may be less than 1 year; for patients with high risk of molecular features combined with M0&T2, M0&T4, LNR 0& M0, LNR median &T3, and LNR high, the survival is predicted less than 3 years; and the survival of patients with M1&T3, M0 and high risk molecular features is less than 5 years. Using multidimensional and complex patient information, this study establishes potential criteria for clinicians to evaluate the survival of patients for colon cancer.

The isolation and identification of Candida glabrata from avian species and a study of the antibacterial activities of Chinese herbal medicine in vitro.

Previously, a fungus was isolated from a diseased pigeon group clinically suspected of being infected with Candida. The fungus was subsequently identified as Candida glabrata using morphology, physiology, biochemistry, and molecular biology testing methods. In the present study, to determine the controlling effects of Chinese herbal medicine for C. glabrata, the bacteriostatic effects of the ethanol extracts Acorus gramineus, Sophora flavescens, Polygonum hydropiper, Cassia obtusifolia, Pulsatilla chinensis, Dandelion, and Cortex phellodendri on C. glabrata in vitro were analyzed. The results showed that the minimum inhibitory concentrations (MIC80) of Cortex phellodendri was 0.25 µg/µL. Meanwhile, that of S. flavescens was 32 µg/µL; C. obtusifolia was 56 µg/µL; A. gramineus and Polygonum hydropiper was 64 µg/µL; and P. chinensis was 112 µg/µL. However, MIC80 for Dandelion was undetectable. In addition, improved drug sensitivity tests revealed that colonies had grown after 24 h in the blank group, as well as the Polygonum hydropiper, P. chinensis, Dandelion, and ethanol groups. The colonies first appeared at the 48-hour point in the other drug-sensitive medium of Chinese herbal medicine. However, no colony growth was found in Cortex phellodendri medium, and the formation of the maximum colony diameter in that group was later than the blank group (e.g., 96 h in the blank group and 120 h in the Chinese herbal medicine group). It was observed that only 17 colony-forming units had grown in 125 µg/µL of the S. flavescens medium, which was significantly different from other groups. Also, the final colony diameter was significantly smaller than that of the other experimental groups. Therefore, it was determined that the A. gramineus, S. flavescens, Polygonum hydropiper, Cassia obtusifolia, P. chinensis, and Cortex phellodendri had certain inhibitory effects on the growth of the C. glabrata. Among those, it was observed that the Cortex phellodendri had the strongest inhibitory effects, followed by the S. flavescens. In the future, these Chinese herbal medicines are expected to be used to treat the fungal infections related to C. glabrata in poultry to improve production performance.

Study on the bacteriostatic action of Chinese herbal medicine on avian Trichosporon.

In this study, a strain of Trichosporon was isolated from white pseudomembranes and ulcers formed on mucous membranes of pigeon bursas and was identified through gene sequencing. Bacteriostatic actions of Acorus gramineus, Sophora flavescens, Polygonum hydropiper, and Chinese herbal mixture on this species were explored in vitro, and the minimum inhibitory concentration of herbal medicines against Trichosporon was determined through microdilution method. Therapeutic effects of herbal medicines on chickens infected by Trichosporon were studied, whose results showed that minimum inhibitory concentration of A. gramineus was 32 µg/µL, that of S. flavescens was 2 µg/µL, that of P. hydropiper was 120 µg/µL, and that of Chinese herbal mixture was 36 µg/µL. Antibacterial effects of S. flavescens were the best. In accordance with animal experiments, therapeutic effects of Chinese herbal medicines on infected chickens were better than those of fluconazole. The mortality rate of the Chinese herbal medicine treatment group was 33.33%, that of the fluconazole treatment group was 46.67%, and that of the Chinese medicine protection group was 23.33%. The longer the time of Chinese medicine treatments was, the better the treatment effects would be. Glutamic oxaloacetylase values of the serum and liver in the Chinese herbal medicine treatment group were both significantly lower than those of the nontreatment group. From the results, it can be seen that A. gramineus, S. flavescens, P. hydropiper, and Chinese herbal mixture have certain inhibitory effects on Trichosporon spp. Chinese herbal medicine protections in advance could reduce Trichosporon infections.