bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of Raman spectroscopy and machine learning for
Candida auris
identification and characterization
Authors: Xue Junjing , Yue Huizhen , Lu Weilai , Li Yanying , Huang Guanghua , Fu Yu Vincent 
Published: October 2024

ABSTRACT

Candida auris,
an emerging fungal pathogen characterized by multidrug resistance and high-mortality nosocomial infections, poses a serious global health threat. However, the precise and rapid identification and characterization of
C. auris
remain a challenge. Here, we employed Raman spectroscopy combined with machine learning to identify
C. auris
isolates and its closely related species as well as to predict antifungal resistance and key virulence factors at the single-cell level. The average accuracy of identification among all
Candida
species was 93.33%, with an accuracy of 98% for the clinically simulated samples. The drug susceptibility of
C. auris
to fluconazole and amphotericin B was 99% and 94%, respectively. Furthermore, the phenotypic prediction of
C. auris
yielded an accuracy of 100% for aggregating cells and 97% for filamentous cells. This proof-of-concept methodology not only precisely identifies
C. auris
at the clade-specific level but also rapidly predicts the antifungal resistance and biological characteristics, promising a valuable medical diagnostic tool to combat this multidrug-resistant pathogen in the future.

IMPORTANCE

Currently, combating
Candida auris
infections and transmission is challenging due to the lack of efficient identification and characterization methods for this species. To address these challenges, our study presents a novel approach that utilizes Raman spectroscopy and artificial intelligence to achieve precise identification and characterization of
C. auris
at the singe-cell level. It can accurately identify a single cell from the four
C. auris
clades. Additionally, we developed machine learning models designed to detect antifungal resistance in
C. auris
cells and differentiate between two distinct phenotypes based on the single-cell Raman spectrum. We also constructed prediction models for detecting aggregating and filamentous cells in
C. auris
, both of which are closely linked to its virulence. These results underscore the merits of Raman spectroscopy in the identification and characterization of
C. auris
, promising improved outcomes in the battle against
C. auris
infections and transmission.
Conserved residues of the immunosuppressive domain of MLV are essential for regulating the fusion-critical SU-TM disulfide bond
Authors: Hogan Victoria A. , Harmon Julia , Cid-Rosas Miguel , Hall Laura R. , Johnson Welkin E. 
Published: October 2024

ABSTRACT

The Env protein of murine leukemia virus (MLV) is the prototype of a large clade of retroviral fusogens, collectively known as gamma-type Envs. Gamma-type Envs are found in retroviruses and endogenous retroviruses (ERVs) representing a broad range of vertebrate hosts. All gamma-type Envs contain a highly conserved stretch of 26-residues in the transmembrane subunit (TM) comprising two motifs, a putative immunosuppressive domain (ISD) and a CX
6
CC motif. Extraordinary conservation of the ISD and its invariant association with the CX
6
CC suggests a fundamental contribution to Env function. To investigate ISD function, we characterized several mutants with single amino acid substitutions at conserved positions in the MLV ISD. A majority abolished infectivity, although we did not observe a corresponding loss in intrinsic ability to mediate membrane fusion. Ratios of the surface subunit (SU) to capsid protein (CA) in virions were diminished for a majority of the ISD mutants, while TM:CA ratios were similar to wild type. Specific loss of SU reflected premature isomerization of the labile disulfide bond that links SU and TM prior to fusion. Indeed, all non-infectious mutants displayed significantly lower disulfide stability than wild-type Env. These results reveal a role for ISD positions 2, 3, 4, 7, and 10 in regulating a late step in entry after fusion peptide insertion but prior to creation of the fusion pore. This implies that the ISD is part of a larger domain, comprising the ISD and CX
6
CC motifs, that is critical for the formation and regulation of the metastable, intersubunit disulfide bond.

IMPORTANCE
The gamma-type Env is a prevalent viral fusogen, found within retroviruses and endogenous retroviruses across vertebrate species and in filoviruses such as Ebolavirus. The fusion mechanism of gamma-type Envs is unique from other Class I fusogens such as those of influenza A virus and HIV-1. Gamma-type Envs contain a hallmark feature known as the immunosuppressive domain (ISD) that has been the subject of some controversy in the literature surrounding its putative immunosuppressive effects. Despite the distinctive conservation of the ISD, little has been done to investigate the role of this region for the function of this widespread fusogen. Our work demonstrates the importance of the ISD for the function of gamma-type Envs in infection, particularly in regulating the intermediate steps of membrane fusion. Understanding the fusion mechanism of gamma-type Envs has broad implications for understanding the entry of extant viruses and aspects of host biology connected to co-opted endogenous gamma-type Envs.
Structures of Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus virions reveal species-specific tegument and envelope features
Authors: Zhen James , Chen Jia , Huang Haigen , Liao Shiqing , Liu Shiheng , Yuan Yan , Sun Ren , Longnecker Richard , Wu Ting-Ting , Zhou Z. Hong 
Published: October 2024

ABSTRACT

Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are classified into the gammaherpesvirus subfamily of
Herpesviridae
, which stands out from its alpha- and betaherpesvirus relatives due to the tumorigenicity of its members. Although structures of human alpha- and betaherpesviruses by cryogenic electron tomography (cryoET) have been reported, reconstructions of intact human gammaherpesvirus virions remain elusive. Here, we structurally characterize extracellular virions of EBV and KSHV by deep learning-enhanced cryoET, resolving both previously known monomorphic capsid structures and previously unknown pleomorphic features beyond the capsid. Through subtomogram averaging and subsequent tomogram-guided sub-particle reconstruction, we determined the orientation of KSHV nucleocapsids from mature virions with respect to the portal to provide spatial context for the tegument within the virion. Both EBV and KSHV have an eccentric capsid position and polarized distribution of tegument. Tegument species span from the capsid to the envelope and may serve as scaffolds for tegumentation and envelopment. The envelopes of EBV and KSHV are less densely populated with glycoproteins than those of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV), representative members of alpha- and betaherpesviruses, respectively. Also, we observed fusion protein gB trimers exist within triplet arrangements in addition to standalone complexes, which is relevant to understanding dynamic processes such as fusion pore formation. Taken together, this study reveals nuanced yet important differences in the tegument and envelope architectures among human herpesviruses and provides insights into their varied cell tropism and infection.

IMPORTANCE
Discovered in 1964, Epstein-Barr virus (EBV) is the first identified human oncogenic virus and the founding member of the gammaherpesvirus subfamily. In 1994, another cancer-causing virus was discovered in lesions of AIDS patients and later named Kaposi’s sarcoma-associated herpesvirus (KSHV), the second human gammaherpesvirus. Despite the historical importance of EBV and KSHV, technical difficulties with isolating large quantities of these viruses and the pleiomorphic nature of their envelope and tegument layers have limited structural characterization of their virions. In this study, we employed the latest technologies in cryogenic electron microscopy (cryoEM) and tomography (cryoET) supplemented with an artificial intelligence-powered data processing software package to reconstruct 3D structures of the EBV and KSHV virions. We uncovered unique properties of the envelope glycoproteins and tegument layers of both EBV and KSHV. Comparison of these features with their non-tumorigenic counterparts provides insights into their relevance during infection.
Acquisition of a multibasic cleavage site does not increase MERS-CoV entry into Calu-3 human lung cells
Authors: Hoffmann Markus , Kleine-Weber Hannah , Graichen Luise , Nehlmeier Inga , Kempf Amy , Moldenhauer Anna-Sophie , Braun Elisabeth , Assiri Abdullah M. , Kirchhoff Frank , Sauter Daniel , Alkharsah Khaled R. , Pöhlmann Stefan 
Published: October 2024

ABSTRACT

Human-to-human transmission of the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) is currently inefficient. However, there is concern that the virus might mutate and thereby increase its transmissibility and thus pandemic potential. The pandemic SARS-CoV-2 depends on a highly cleavable furin motif at the S1/S2 site of the viral spike (S) protein for efficient lung cell entry, transmission, and pathogenicity. Here, by employing pseudotyped particles, we investigated whether augmented cleavage at the S1/S2 site also increases MERS-CoV entry into Calu-3 human lung cells. We report that polymorphism T746K at the S1/S2 cleavage site or optimization of the furin motif increases S protein cleavage but not lung cell entry. These findings suggest that, unlike what has been reported for SARS-CoV-2, a highly cleavable S1/S2 site might not augment MERS-CoV infectivity for human lung cells.

IMPORTANCE
The highly cleavable furin motif in the spike protein is required for robust lung cell entry, transmission, and pathogenicity of SARS-CoV-2. In contrast, it is unknown whether optimization of the furin motif in the spike protein of the pre-pandemic MERS-CoV increases lung cell entry and allows for robust human–human transmission. The present study indicates that this might not be the case. Thus, neither a naturally occurring polymorphism that increased MERS-CoV spike protein cleavage nor artificial optimization of the cleavage site allowed for increased spike-protein-driven entry into Calu-3 human lung cells.
A near-complete genome of the uncultured
Staphylococcus aureus
phage COMBAT-CF_PAR1 isolated from the lungs of an infant with cystic fibrosis
Authors: Agudelo-Romero Patricia , Caparros-Martin Jose A. , Sharma Abhinav , Saladié Montserrat , Sly Peter D. , Stick Stephen M. , O´Gara Fergal ,   COMBAT study group
Published: October 2024

ABSTRACT

In cystic fibrosis, bacteria–bacteriophage interaction in the lower airways is poorly understood. We present the near-complete genome of the uncultured Siphovirus-like bacteriophage,
Staphylococcus aureus
phage COMBAT-CF_PAR1, isolated from the lower airways. The genome spans 41,510 bp with 33.45% guanine–cytosine content and contains 65 open reading frames.
Metagenomic sequencing of CRISPRs as a new marker to aid in personal identification with low-biomass samples
Authors: Toyomane Kochi , Kimura Yuri , Fukagawa Takashi , Yamagishi Takayuki , Watanabe Ken , Akutsu Tomoko , Asahi Ai , Kubota Satoshi , Sekiguchi Kazumasa 
Published: October 2024

ABSTRACT

The high specificity of the human skin microbiome is expected to provide a new marker for personal identification. Metagenomic sequencing of clustered regularly interspaced short palindromic repeats (CRISPRs), which we call metaCRISPR typing, was shown to achieve personal identification accurately. However, the intra-individual variability observed in previous studies, which may be due to poor DNA yields from skin samples, has resulted in non-reproducible results. Furthermore, whether metaCRISPR typing can assist in the forensic human DNA analysis of low-biomass samples, from which the information obtained is insufficient, is unknown. In the present study, we sequenced serially diluted control streptococcal CRISPRs cloned into plasmids to determine the minimum copy number required to obtain reproducible results from metaCRISPR typing. We found that at least 10
2
copies of CRISPRs are necessary to obtain reproducible results. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA typing. When the DNA extracted from the skin swabs was diluted, no information was obtained from six out of eight samples by human DNA typing. On the other hand, beta diversity indices of spacer sequences compared with reference samples were below 0.8 for three out of six samples, for which no information was obtained from human DNA analysis, indicating that the spacers observed in these samples were similar to those in the references. These results indicate that metaCRISPR typing may contribute to the identification of individuals from whom the samples were obtained, even in cases where human DNA yields are insufficient to perform human DNA analysis.

IMPORTANCE
Previous studies have developed new personal identification methods utilizing personal differences in the skin microbiome. However, intra-individual diversity of skin microbiome may preclude the application of microbiome-based personal identification. Moreover, no study has compared microbiome-based personal identification and practical human DNA analysis. Here, we revealed that the results of metaCRISPR typing, a previously developed microbiome-based personal identification method, are stable if the copy number of the marker gene is sufficient. We then analyzed the skin swab samples using both metaCRISPR typing and human DNA analysis. Our results indicate that metaCRISPR typing may provide additional information for personal identification using low-biomass samples that cannot be used for conventional human DNA analysis.
False-positive results for seasonal coronavirus infections on using the FilmArray Pneumonia Panel
Authors: Park Kuenyoul , Park Bosung , Won Eun Jeong , Sung Heungsup , Kim Mi-Na 
Published: October 2024

ABSTRACT

The FilmArray Pneumonia Panel (FilmArrayPN; bioMérieux) was tagged for potential false-positive seasonal coronavirus results, possibly caused by non-specific amplification or cross-reactivity with human genomic DNA. This study was conducted to determine the real-world false-positive rate of seasonal coronavirus infections using FilmArrayPN at a tertiary care hospital. We retrospectively reviewed FilmArrayPN results to detect lower respiratory tract pathogens from July 2023 to April 2024. Allplex Respiratory Panel (AllplexRP; Seegene) and/or FilmArray Respiratory Panel (FilmArrayRP) results for the same patient were considered as reference results. In the last month of the study period, all positive samples in FilmArrayPN were confirmed by AllplexRP and pan-coronavirus targeted PCR (pan-CoV PCR). A total of 2,120 specimens, including 1,847 expectorated sputum and 273 bronchial aspirate (BA) samples, were tested by FilmArrayPN. Of these, 161 (8.7%) sputum and 7 (2.6%) BA specimens from 152 patients were positive for coronavirus targets in FilmArrayPN. Of the 122 cases also tested by AllplexRP and/or FilmArrayRP, 106 specimens (86.9%) were coronavirus-negative, except for 9 OC43-positive, 6 229E-positive, and 1 NL63-positive. Notably, 83.1% of the 106 cases that tested negative in the other tests had melting temperatures above 83℃. A total of 61 specimens that tested positive for coronavirus in FilmArrayPN but negative in AllplexRP were confirmed to be negative in pan-CoV PCR. The coronavirus positivity of 7.8% in the FilmArrayPN resulted in 5% of samples being potentially misreported as false-positives. This report thus highlights the need for continuous monitoring of melting temperatures to avoid potential false-positives.

IMPORTANCE
The FilmArray Pneumonia Panel (FilmArrayPN; bioMérieux) was tagged for potential false-positive seasonal coronavirus results, possibly caused by non-specific amplification or cross-reactivity with human genomic DNA. FilmArrayPN results were retrospectively reviewed from July 2023 to May 2024. Of 2,120 tested specimens, 168 specimens from 152 patients were positive for coronavirus targets in FilmArrayPN. Of the 122 cases also tested by Allplex Respiratory Panel and/or FilmArray Respiratory Panel, 106 specimens (86.9%) were coronavirus-negative. Notably, 83.1% of the 106 cases that tested negative in the other tests had melting temperatures above 83℃. A total of 61 specimens that tested positive for coronavirus in FilmArrayPN but negative in Allplex were confirmed to be negative in pan-coronavirus targeted PCR. The coronavirus positivity of 7.8% in the FilmArrayPN resulted in 5% of samples being potentially misreported as false-positives. This report highlights the need for continuous monitoring of melting temperatures to avoid potential false-positives.
Unveiling environmental transmission risks: comparative analysis of azole resistance in
Aspergillus fumigatus
clinical and environmental isolates from Yunnan, China
Authors: Gong Jianchuan , Huang Jiarui , Liu Yongju , Zhang Ying , Gao Yuhong 
Published: October 2024

ABSTRACT

Azole resistance in
Aspergillus fumigatus
poses a significant clinical challenge globally. Our previous epidemiological analysis revealed a remarkably high frequency (~80%) of azole-resistant
A. fumigatus
in Yunnan’s greenhouse environments, prompting increased local and regional research for targeted control strategies. In this study, we analyzed 94 clinical
A. fumigatus
isolates from Yunnan, comparing their susceptibility profiles and genotypic characteristics with environmental strains previously isolated. While the overall frequency of azole resistance in clinical isolates was lower than that in environmental samples, a significant prevalence of cross-resistance, with varying resistance patterns based on minimum inhibitory concentration (MIC) levels was observed, which exceeded rates in other regions of China. Specific mutation combinations in the
cyp51A
gene were linked to elevated MIC values in clinical and/or environmental samples, while some resistant strains with wild-type
cyp51A
remain unexplained, indicating a need for further investigation into their resistance mechanisms. The differences in unique genetic elements and the distinct genetic differentiation observed between clinical and environmental isolates can be attributed to Yunnan’s unique geomorphology and potential genotype importation from other provinces and abroad. Extensive allele exchanges and sharing contributed to the selection of azole-resistant clinical isolates, suggesting a common environmental origin, and the transmission routes of local drug-resistant strains cannot be excluded. These findings emphasize the imperative for regional and targeted surveillance to monitor resistance trends and guide effective antifungal therapy, and management strategies to mitigate invasive aspergillosis risk in this region.

IMPORTANCE

Azole resistance in
Aspergillus fumigatus
is a major global health concern, with particularly high rates (~80%) observed in Yunnan’s greenhouse environments. This study compares azole resistance in 94 clinical isolates from Yunnan with environmental strains, revealing lower clinical resistance but significant cross-resistance and distinct resistance patterns. Specific mutations in the
cyp51A
gene were associated with elevated minimum inhibitory concentration values, though some resistant strains had wild-type
cyp51A
, highlighting the need for further research. The unique genetic profiles and potential external genotype influences in Yunnan emphasize the need for targeted regional surveillance. Effective monitoring and control strategies are essential to manage and mitigate the risk of invasive aspergillosis.
Key performance evaluation of commercialized multiplex rRT-PCR kits for respiratory viruses: implications for application and optimization
Authors: Feng Wanyu , Chen Yuqing , Han Yanxi , Diao Zhenli , Zhao Zihong , Zhang Yuanfeng , Huang Tao , Ma Yu , Li Ziqiang , Jiang Jian , Li Jing , Li Jinming , Zhang Rui 
Published: October 2024

ABSTRACT

Respiratory tract infections (RTIs) caused by viruses are prevalent and significant conditions in clinical settings. Accurate and effective detection is of paramount importance in the diagnosis, treatment, and prevention of viral RTIs. With technological advancements, multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assays have been developed and extensively adopted for the diagnosis of viral RTIs. Given the potential challenges in the detection performance of multiplex assays, this study evaluated the analytical sensitivity and competitive interference of the six most commonly used multiplex rRT-PCR kits for detection of respiratory viruses in China. The results revealed that the limits of detection were variable across the viruses and kits. Most of the evaluated multiplex kits demonstrated comparable or enhanced analytical sensitivity compared with singleplex kits for clinically significant viruses, including human adenovirus (HAdV)-3, HAdV-7, Omicron BA.5, H1N1pdm09, H3N2, B/Victoria, respiratory syncytial virus subtype A, and respiratory syncytial virus subtype B, whereas multiplex kits showed relatively less analytical sensitivity for human rhinovirus-B72, human metapneumovirus-A2, parainfluenza virus (PIV)-1, and PIV-3. In addition, most multiplex kits successfully identified co-infections when one analyte was present at a low concentration and another analyte was present at a high concentration.

IMPORTANCE
The complexity and severity of viral respiratory tract infections (RTIs) emphasize the pivotal role of precise diagnosis for viral RTIs in guiding effective public health responses and ensuring appropriate medical interventions, given the substantial population at risk. This study highlights the necessity and importance of evaluating the analytical validity of multiplex real-time reverse transcription polymerase chain reaction assays, offering valuable insights into their optimization and application.
Effectiveness of paediatric asthma hubs: a clinical pilot study
Authors: Hakizimana Ali , Lo David K H , Roland Damian , Rai Vinayak K , Danvers Lesley , Rowlands Rachel , Ahmed Molla Imaduddin , Herzallah Reeta , Gaillard Erol A 
Published: October 2024

BackgroundChildren and young people (CYP) with asthma in the UK are at higher risk of poor outcomes compared with other high-income European countries due to factors including poor access to high-quality asthma reviews, diagnostic testing and inconsistent postattack reviews. The Leicester Integrated Care Board funded the first UK pilot asthma hub for CYP, to investigate the feasibility and effectiveness of hubs, in providing postattack reviews along with providing asthma education, the opportunity to carry out diagnostic lung function tests and optimise treatment.MethodsClinical pilot study including CYP aged 4–17 years referred to the hub with uncontrolled asthma or postattack from November 2021 to April 2022. CYP received a structured clinical assessment including National Institute for Health and Care Excellence (NICE) first-line diagnostic investigations for asthma including spirometry, bronchodilator reversibility (BDR) and fraction of exhaled nitric oxide (FeNO).ResultsOf 312 CYP referred (mean age 8.6±3.2 years; 42% women), 266 (85.3%) attended their appointment. Median time from referral to review was 2 days (IQR 1–3). Three CYP (1.1%) were severely unwell at review and required further hospital treatment. In the 231 CYP who completed first-line tests, asthma was confirmed for 73 (31.6%) based on NICE diagnostic criteria for CYP. Twenty-two per cent of children with normal baseline spirometry had ≥12% BDR.ConclusionPaediatric asthma hubs are a feasible model of care to deliver CYP postasthma attack reviews and identify high-risk patients requiring further treatment. Spirometry, BDR and FeNO testing allowed diagnostic confirmation in a significant proportion of CYP.