Analysis of the molecular basis for the non-amylolytic and non-proteolytic nature of Aspergillus vadensis CBS 113365.

Aspergillus vadensis CBS 113365, a close relative of A. niger, has been suggested as a more favourable alternative for recombinant protein production as it does not acidify the culture medium and produces very low levels of extracellular proteases. The aim of this study was to investigate the underlying cause of the non-amylolytic and non-proteolytic phenotype of A. vadensis CBS 113365. Our results demonstrate that the non-functionality of the amylolytic transcription factor AmyR in A. vadensis CBS 113365 is primarily attributed to the lack of functionality of its gene's promoter sequence. In contrast, a different mechanism is likely causing the lack of PrtT activity, which is the main transcriptional regulator of protease production. The findings presented here not only expand our understanding of the genetic basis behind the distinct characteristics of A. vadensis CBS 113365, but also underscore its potential as a favourable alternative for recombinant protein production.

Comparison of consistency in acute stroke: Automated ASPECT score software tools and manual consensus scores.

PURPOSE: The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) is a standardized semi-quantitative scoring system. It is widely used to assess the extent of acute ischemic stroke (AIS). We evaluated the consistency of three automatic software packages with the overall and region-specific manual ASPECTS scores of AIS patients. METHODS: Retrospectively, we gathered patients who presented with stroke symptoms between February 2019 and June 2022, and 174 cases were eventually included in the trial. Two radiologists reviewed the NCCT images independently; After four weeks, the same two radiologists began randomly reviewing the DWI images, discussed different scores and give consistent results as ground truth. RESULTS: Median ASPECTS of the expert consensus reading was 7 (5-9). Good to excellent correlation of ASPECTS total scores among the three software tools (0.70, 0.74 and 0.83). Correlation among ground truth and Rapid-ASPECTS, RealNow-ASPECTS, ShuKun-ASPECTS (ICC = 0.51, Cronbach's α = 0.53), (ICC = 0.60, Cronbach's α = 0.70) and (ICC = 0.52, Cronbach's α = 0.64) respectively. The AUCs for Rapid, RealNow and ShuKun were 0.61, 0.67, and 0.62 respectively. The region-specific results showed a poor to good correlation. The correlations between the non-dominant and dominant cerebral hemispheres and the ground truth were statistically different (P < 0.05). CONCLUSIONS: Overall, the scoring consistency between the three automated scoring software and the ground truth is comparable, with RealNow-ASPECTS being no less consistent and effective than Rapid-ASPECTS and ShuKun-ASPECTS, and even better than both. But the consistency grade that still is developable.

Heterologous protein production in filamentous fungi.

Filamentous fungi are able to produce a wide range of valuable proteins and enzymes for many industrial applications. Recent advances in fungal genomics and experimental technologies are rapidly changing the approaches for the development and use of filamentous fungi as hosts for the production of both homologous and heterologous proteins. In this review, we highlight the benefits and challenges of using filamentous fungi for the production of heterologous proteins. We review various techniques commonly employed to improve the heterologous protein production in filamentous fungi, such as strong and inducible promoters, codon optimization, more efficient signal peptides for secretion, carrier proteins, engineering of glycosylation sites, regulation of the unfolded protein response and endoplasmic reticulum associated protein degradation, optimization of the intracellular transport process, regulation of unconventional protein secretion, and construction of protease-deficient strains. KEY POINTS: • This review updates the knowledge on heterologous protein production in filamentous fungi. • Several fungal cell factories and potential candidates are discussed. • Insights into improving heterologous gene expression are given.

Screening Children's Intellectual Disabilities with Phonetic Features, Facial Phenotype and Craniofacial Variability Index.

BACKGROUND: Intellectual Disability (ID) is a kind of developmental deficiency syndrome caused by congenital diseases or postnatal events. This syndrome could be intervened as soon as possible if its early screening was efficient, which may improve the condition of patients and enhance their self-care ability. The early screening of ID is always achieved by clinical interview, which needs in-depth participation of medical professionals and related medical resources. METHODS: A new method for screening ID has been proposed by analyzing the facial phenotype and phonetic characteristic of young subjects. First, the geometric features of subjects' faces and phonetic features of subjects' voice are extracted from interview videos, then craniofacial variability index (CVI) is calculated with the geometric features and the risk of ID is given with the measure of CVI. Furthermore, machine learning algorithms are utilized to establish a method for further screening ID based on facial features and phonetic features. RESULTS: The proposed method using three feature sets, including geometric features, CVI features and phonetic features was evaluated. The best performance of accuracy was closer to 80%. CONCLUSIONS: The results using the three feature sets revealed that the proposed method may be applied in a clinical setting in the future after continuous improvement.

A lightweight CNN and Transformer hybrid model for mental retardation screening among children from spontaneous speech.

Mental retardation (MR) is a group of mental disorders characterized by low intelligence and social adjustment difficulties. Early diagnosis is beneficial for the timely intervention of children with MR to ease the degree of disability. Children with MR always have impaired speech functions compared to normal children, which is significant for clinical diagnosis. On the basis of this, our study proposes a spontaneous speech-based framework (MT-Net) for screening MR, which merges mobile inverted bottleneck convolutional blocks (MBConv) and visual Transformer blocks. MT-Net takes log-mel spectrograms converted from raw interview speech as data source, and utilizes MBConv and visual Transformer to learn low-level and high-level features well. In addition, SpecAugment, a data augmentation strategy, has been used to expand our audio dataset to further enhance the performance of MT-Net. The experimental results show that our proposed MT-Net outperforms Transformer networks (ViT) and convolutional neural networks (ResNet18, MobileNetV2, EfficientNetV2), achieving accuracy of 91.60% after using SpecAugment. Our proposed MT-Net has fewer parameters, low computing consumption and high prediction accuracy, which is expected to be an auxiliary screening tool for MR.

Two stream Non-Local CNN-LSTM network for the auxiliary assessment of mental retardation.

At present, the assessment of mental retardation is mainly based on clinical interview, which requires the participation of experienced psychiatrist and is laborious. Studies have shown that there are correlations between mental retardation and abnormal behaviors (such as, hyperkinetic, tics, stereotypes, etc.). On the basis of this fact, a two stream Non-Local CNN-LSTM network has been proposed to learn the features of upper body behavior and facial expression of patients, thus, to achieve the preliminary screening of mental retardation. Specifically, RGB and optical flow are extracted separately from interview videos, and a two stream network based on contribution mechanism is designed to effectively fuse the information of two kinds of images, which may update the network in a new approach of alternating iteration training to find the optimal model. Besides, by introducing non-local mechanism and adopting it to the network, the global feature sensing can be established more effectively to reduce the background interference for video clip in a short time zone. Experiments on clinical video dataset show that the performance of proposed model is better than other prevalent deep learning methods of behavioral feature learning, the accuracy reaches 89.15% in basic experiment, and is further improved to 89.52% in the supplementary experiment. Furthermore, the experimental results show that this method still has a lot of room for improvement. In general, our work indicates that the proposed model has potential value for the clinical diagnosis and screening of mental retardation.

Germline and somatic mutation profile in Cancer patients revealed by a medium-sized pan-Cancer panel.

Gene mutation detection and the resulted precision-medicine therapy is transforming clinical practice. Here, we report the use of a custom-developed, medium-sized, pan-cancer probe panel for the detection of somatic and germline mutations. We used a hybridization capture-based NGS assay for targeted deep sequencing of all exons and selected introns of 181 key cancer driver genes, covering both inherited risks and somatic mutations. We performed paired-variant calling on tumor samples and their matched normal samples. We processed clinical patient samples of formalin-fixed, paraffin embedded tumors (FFPE samples) and cell-free peripheral blood (cfDNA samples). We found germline mutations of inherited cancer risk at 9%; and discovered a novel germline mutation in BRCA1. Somatic mutation rate in driver genes is at 73.1%, much higher than previously reported. On recommending precision-medicine therapeutics, we achieved 91.6% for patients with FFPE samples.

Molecular mutation characteristics of mismatch and homologous recombination repair genes in gastrointestinal cancer.

Gastrointestinal cancer is one of the most common types of cancer with high mortality rates. Mutations in several genes are reportedly involved in the progression of gastrointestinal cancer, including tumor protein 53 (TP53), APC regulator of WNT signaling pathway (APC), KRAS proto-oncogene GTPase (KRAS) and erb-b2 receptor tyrosine kinase 2 (ERBB2). Most notably, there are numerous mutations in DNA repair genes, including mismatch repair (MMR) and homologous recombination (HR) genes. The focus of the present study was to investigate the effects of MMR and HR gene mutations on genomic instability in gastrointestinal cancer. Using targeted capture and massively parallel genomic sequencing, 137 gastrointestinal cancer patients were analyzed for somatic single-nucleotide variants (SNVs) and insertion-deletion (indel) mutations in the exon regions of 183 cancer driver genes, including 4 MMR genes [MutL homolog MLH1, MLH2, MLH6 and PMS1 homolog 2, mismatch repair system component (PMS2)] and 15 HR genes [BRCA1 DNA repair associated (BRCA1), BRCA2 DNA repair associated (BRCA2), ATM serine/threonine kinase (ATM), phosphatase and tensin homolog, BLM RecQ like helicase, FA complementation group A, FA complementation group C, FA complementation group D2, FA complementation group E, FA complementation group F, FA complementation group G, nibrin, partner and localizer of BRCA2 and Werner syndrome RecQ like helicase]. A number of frequently mutated genes, including but not limited to, mechanistic target of rapamycin kinase, neurofibromin 1, APC and, in particular, DNA repair genes, including PMS2, ATM and BRCA2, were identified. Frequency analysis was performed based on the SNVs and indels in the 183 genes to indirectly indicate the relative status of genomic instability in each patient. Correlation analysis suggested that MMR and HR gene mutations directly affected the count of SNVs and indels. Overall, 56 of the gastrointestinal cancer patients (40%) were found to have an inactivation mutation (stopgain/frameshift/splicing) in one or more of the four MMR genes, whereas 112 patients (82%) harbored at least one HR gene inactivation mutation. In addition, patients with MMR or HR inactivation variants had more SNVs and indels compared with patients with no such mutations. No other clinical characteristics (including sex and age) appeared to have a statistically significant impact. Further analysis indicated that different MMR or HR genes exerted distinct effects on genomic instability. The results obtained in the current study may lay a foundation for investigations into the tumorigenic process and for the development of novel therapeutic strategies for the treatment of gastrointestinal cancer.

Validity of an NGS-based multiple gene panel in identifying actionable mutations for patients with NSCLC in a Chinese hospital.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. A number of targeted therapies have been approved for clinical use or are in clinical trials. Next generation sequencing (NGS) is widely applied in the identification of actionable genomic alterations and enables personalized cancer therapy for patients. Several multiple-gene panels are available in China for the practice of precision medicine-based cancer therapy. However, the efficiency of these panels requires evaluation. The current study investigated 23 NSCLC samples using a custom designed panel of complete coding regions of ~180 cancer driver genes (FD-180) and whole exome sequencing for control samples, obtained from white blood cell samples. The results obtained suggested that actionable mutations with available targeted therapeutic options were identified in 69.6% of cases, including 60.9% of therapeutic targets recommended by the National Comprehensive Cancer Network guidelines. Furthermore, 8.7% of patients had a gene mutation that potentially qualified them for clinical trials or associated off-label therapies. As such, the results obtained in the current study demonstrated the reliability of the targeted NGS panel and its potential use for identifying actionable gene alterations and designing personalized therapies for patients with NSCLC.

Genomic alterations in gastric cancers discovered via whole-exome sequencing.

BACKGROUND: Gastric cancer (GC) ranks the second in mortality rate among all cancers. Metastases account for most of the deaths in GC patients. Yet our understanding of GC and its metastasis mechanism is still very limited. METHODS: We performed 20 whole-exome sequencing (WES) on 5 typical metastatic gastric adenocarcinoma (GAC) patients with lymph node metastasis. We compared both the primary tumors to their metastatic lymph nodes, and a specific analysis pipeline was used to detect single nucleotide variants (SNVs), small insertions/deletions (indels) and copy number variants (CNVs). RESULTS: (1) We confirmed 30 candidate mutations in both primary and lymph nodes tissues, and other 7 only in primary tumors. (2) Copy number gains were observed in a large section of 17q12-21, as well as copy number losses in regions containing CDKN2A and CDKN2B in both primary and lymph nodes tissues. CONCLUSIONS: Our results provide preliminary insights in the molecular mechanisms of GC initiation, development, and metastatic progression. These results need to be validated through large-scale studies.

rAAV/ABAD-DP-6His attenuates oxidative stress-induced injury of PC12 cells.

Our previous studies have revealed that amyloid ß (Aß)-binding alcohol dehydrogenase (ABAD) decoy peptide antagonizes Aß42-induced neurotoxicity. However, whether it improves oxidative stress injury remains unclear. In this study, a recombinant adenovirus constitutively secreting and expressing Aß-ABAD decoy peptide (rAAV/ABAD-DP-6His) was successfully constructed. Our results showed that rAAV/ABAD-DP-6His increased superoxide dismutase activity in hydrogen peroxide-induced oxidative stress-mediated injury of PC12 cells. Moreover, rAAV/ABAD-DP-6His decreased malondialdehyde content, intracellular Ca(2+) concentration, and the level of reactive oxygen species. rAAV/ABAD-DP-6His maintained the stability of the mitochondrial membrane potential. In addition, the ATP level remained constant, and apoptosis was reduced. Overall, the results indicate that rAAV/ABAD-DP-6His generates the fusion peptide, Aß-ABAD decoy peptide, which effectively protects PC12 cells from oxidative stress injury induced by hydrogen peroxide, thus exerting neuroprotective effects.

Molecular characterization of a strawberry FaASR gene in relation to fruit ripening.

BACKGROUND: ABA-, stress- and ripening-induced (ASR) proteins have been reported to act as a downstream component involved in ABA signal transduction. Although much attention has been paid to the roles of ASR in plant development and stress responses, the mechanisms by which ABA regulate fruit ripening at the molecular level are not fully understood. In the present work, a strawberry ASR gene was isolated and characterized (FaASR), and a polyclonal antibody against FaASR protein was prepared. Furthermore, the effects of ABA, applied to two different developmental stages of strawberry, on fruit ripening and the expression of FaASR at transcriptional and translational levels were investigated. METHODOLOGY/PRINCIPAL FINDINGS: FaASR, localized in the cytoplasm and nucleus, contained 193 amino acids and shared common features with other plant ASRs. It also functioned as a transcriptional activator in yeast with trans-activation activity in the N-terminus. During strawberry fruit development, endogenous ABA content, levels of FaASR mRNA and protein increased significantly at the initiation of ripening at a white (W) fruit developmental stage. More importantly, application of exogenous ABA to large green (LG) fruit and W fruit markedly increased endogenous ABA content, accelerated fruit ripening, and greatly enhanced the expression of FaASR transcripts and the accumulation of FaASR protein simultaneously. CONCLUSIONS: These results indicate that FaASR may be involved in strawberry fruit ripening. The observed increase in endogenous ABA content, and enhanced FaASR expression at transcriptional and translational levels in response to ABA treatment might partially contribute to the acceleration of strawberry fruit ripening.

Expression and regulation of the early auxin-responsive Aux/IAA genes during strawberry fruit development.

The plant hormone auxin transcriptionally activates Aux/IAA genes. Auxin plays an important role in regulating fruit growth and ripening of strawberry and Aux/IAA genes have been extensively studied in Arabidopsis, rice and tomato, but little information is available on strawberry fruit. In the present work, two full-length of early auxin-responsive Aux/IAA genes, termed FaAux/IAA1 and FaAux/IAA2 respectively, were isolated and characterized from strawberry fruit. Moreover, the expression profiles of two FaAux/IAA genes during fruit development, and the effect of naphthalene acetic acid (NAA) on their expressions of fruits at two different developmental stages were also investigated. The results showed that the levels of FaAux/IAA1 and FaAux/IAA2 transcripts were very high at early stage of fruit development, and decreased sharply at ripening stage (after white stage). In addition, NAA applied at the stage of large green and white fruit obviously increased the accumulations of FaAux/IAA1 and FaAux/IAA2 transcripts. These data suggested that the expressions of both FaAux/IAA1 and FaAux/IAA2 genes were likely to be involved in early fruit development, and the enhancement of FaAux/IAAs transcripts might be attributed at least or partially to auxin-induced fruit growth and delayed fruit ripening of strawberry.