Steroid Hormone Function Controls Non-competitive Plasmodium Development in Anopheles.

Transmission of malaria parasites occurs when a female Anopheles mosquito feeds on an infected host to acquire nutrients for egg development. How parasites are affected by oogenetic processes, principally orchestrated by the steroid hormone 20-hydroxyecdysone (20E), remains largely unknown. Here we show that Plasmodium falciparum development is intimately but not competitively linked to processes shaping Anopheles gambiae reproduction. We unveil a 20E-mediated positive correlation between egg and oocyst numbers; impairing oogenesis by multiple 20E manipulations decreases parasite intensities. These manipulations, however, accelerate Plasmodium growth rates, allowing sporozoites to become infectious sooner. Parasites exploit mosquito lipids for faster growth, but they do so without further affecting egg development. These results suggest that P. falciparum has adopted a non-competitive evolutionary strategy of resource exploitation to optimize transmission while minimizing fitness costs to its mosquito vector. Our findings have profound implications for currently proposed control strategies aimed at suppressing mosquito populations.

A male steroid controls female sexual behaviour in the malaria mosquito.

Insects, unlike vertebrates, are widely believed to lack male-biased sex steroid hormones1. In the malaria mosquito Anopheles gambiae, the ecdysteroid 20-hydroxyecdysone (20E) appears to have evolved to both control egg development when synthesized by females2 and to induce mating refractoriness when sexually transferred by males3. Because egg development and mating are essential reproductive traits, understanding how Anopheles females integrate these hormonal signals can spur the design of new malaria control programs. Here we reveal that these reproductive functions are regulated by distinct sex steroids through a sophisticated network of ecdysteroid-activating/inactivating enzymes. We identify a male-specific oxidized ecdysteroid, 3-dehydro-20E (3D20E), which safeguards paternity by turning off female sexual receptivity following its sexual transfer and activation by dephosphorylation. Notably, 3D20E transfer also induces expression of a reproductive gene that preserves egg development during Plasmodium infection, ensuring fitness of infected females. Female-derived 20E does not trigger sexual refractoriness but instead licenses oviposition in mated individuals once a 20E-inhibiting kinase is repressed. Identifying this male-specific insect steroid hormone and its roles in regulating female sexual receptivity, fertility and interactions with Plasmodium parasites suggests the possibility for reducing the reproductive success of malaria-transmitting mosquitoes.

protoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.

CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

Long-chain polyunsaturated fatty acids influence colorectal cancer progression via the interactions between the intestinal microflora and the macrophages.

The metabolism of long-chain polyunsaturated fatty acids (LCPUFAs) is closely associated with the risk and progression of colorectal cancer (CRC). This paper aims to investigate the role of LCPUFA in the crosstalk between intestinal microflora and macrophages, as well as the effects of these three parties on the progression of CRC. The metabolism and function of LCPUFA play important roles in regulating the composition of the human gut microflora and participating in the regulation of inflammation, ultimately affecting macrophage function and polarization, which is crucial in the tumor microenvironment. The effects of LCPUFA on cellular interactions between the two species can ultimately influence the progression of CRC. In this review, we explore the molecular mechanisms and clinical applications of LCPUFA in the interactions between intestinal microflora and intestinal macrophages, as well as its significance for CRC progression. Furthermore, we reveal the role of LCPUFA in the construction of the CRC microenvironment and explore the key nodes of the interactions between intestinal flora and intestinal macrophages in the environment. It provides potential targets for the metabolic diagnosis and treatment of CRC.

Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease.

The protozoan Trypanosoma cruzi almost invariably establishes life-long infections in humans and other mammals, despite the development of potent host immune responses that constrain parasite numbers. The consistent, decades-long persistence of T. cruzi in human hosts arises at least in part from the remarkable level of genetic diversity in multiple families of genes encoding the primary target antigens of anti-parasite immune responses. However, the highly repetitive nature of the genome-largely a result of these same extensive families of genes-have prevented a full understanding of the extent of gene diversity and its maintenance in T. cruzi. In this study, we have combined long-read sequencing and proximity ligation mapping to generate very high-quality assemblies of two T. cruzi strains representing the apparent ancestral lineages of the species. These assemblies reveal not only the full repertoire of the members of large gene families in the two strains, demonstrating extreme diversity within and between isolates, but also provide evidence of the processes that generate and maintain that diversity, including extensive gene amplification, dispersion of copies throughout the genome and diversification via recombination and in situ mutations. Gene amplification events also yield significant copy number variations in a substantial number of genes presumably not required for or involved in immune evasion, thus forming a second level of strain-dependent variation in this species. The extreme genome flexibility evident in T. cruzi also appears to create unique challenges with respect to preserving core genome functions and gene expression that sets this species apart from related kinetoplastids.

Clone and Function Verification of the OPR gene in Brassica napus Related to Linoleic Acid Synthesis.

BACKGROUND: Fatty acid composition and content affect rapeseed oil quality. Fatty acid synthesis-related genes in rapeseed have been studied globally by researchers. Nevertheless, rapeseed oil is mainly composed of seven different fatty acids (FA), and each fatty acid was regulated by different genes. Furthermore, different FA affect each other, which needs continuous and in-depth research to obtain more clear results in Brassica napus. RESULTS: In this paper, broad-scale miRNA expression profiles were constructed and 21 differentially expressed miRNAs were detected. GO enrichment analysis showed that most up-regulated proteins were involved in transcription factor activity and catalytic activity. KEGG pathway enrichment analysis indicated that 20 pathways involving 36 target genes were enriched, of which the bna00592 pathway may be involved in fatty acid metabolism. The results were verified using a quantitative real-time PCR (RT-qPCR) analysis, we found that the target gene of bna-miR156b > c > g was the OPR (12-oxo-phytodienoic acid reductase). Four copies of OPR gene were found, and the over-expression vectors (pCAMBIA1300-35 s-OPR and pCAMBIA1300-RNAi-OPR) were constructed to verify their functions. In T1 and T2 generation, the content of linoleic acid (LA) increased significantly in OE but deceased in OPRi. CONCLUSIONS: This is the first study to provide four copies of the OPR gene that regulates LA metabolism, can be used for the molecular mechanism of LA and optimizing fatty acid profiles in oilseed for breeding programs.

Establishment of a co-analysis system for personal identification and body fluid identification: a preliminary report.

Analysis of genetic markers can provide clues for case investigation. Short tandem repeat (STR) detection and analysis are widely used for both personal identification and parentage testing. However, DNA analysis currently cannot provide sufficient information for body fluid identification. Tissue or cell sources of samples can be identified by detecting body fluid-specific mRNA markers, which have been studied thoroughly. Integrating STR profiling and mRNA expression patterns can provide more information than conventional methods for investigations and the reconstruction of crime scenes; this can be achieved by DNA/RNA co-extraction technology, which is economical, efficient, and suitable for low-template samples. Here, we propose a co-analysis system based on the PowerPlex 16 kit. This system can simultaneously amplify 25 markers, including 15 STRs, one non-STR amelogenin, and nine mRNA markers (three blood-specific, two saliva-specific, two semen-specific, and two housekeeping gene markers). The specificity and sensitivity of the co-analysis system were determined and aged and degraded samples were used to validate the stability of the co-analysis system. Finally, different DNA/RNA ratios and various carriers were evaluated. The results showed that the DNA/RNA co-analysis system correctly identified different types of body fluid stains. The STR profiles obtained using the co-analysis system were identical to those obtained using the PP16 kit, which demonstrates that the mRNA primers used did not affect STR profiling. Complete STR and mRNA profiles could be obtained from 1/8 portions of buccal swabs, 1/16 portions of swabs of blood and semen samples, 0.1 cm2 of blood samples, 0.25 cm2 of semen samples, and 1.0 cm2 saliva samples. Additionally, our findings indicate that complete STR and mRNA profiles can be obtained with this system from blood and semen samples when the DNA/RNA ratio is 1:1/32. This study suggests that the co-analysis system could be used for simultaneous personal identification and body fluid identification.

An insertion and deletion mutant of adenovirus in Muscovy ducks.

Duck adenovirus 3 (DuAdV-3; strain HB) was isolated and sequenced. The genome of the Muscovy-duck-origin virus contains a 54-bp insertion in pVIII, a 3-bp deletion in the overlap region of 100K, 22K, and 33K, a 42-bp deletion at the junction of ORF64 and ORF67, and a 715-bp deletion in right noncoding region of the genome. Notably, HB has a strikingly shorter right inverted terminal repeat (ITR) of 50 bp, whereas all other DuAdV-3 isolates have a 721-bp ITR. These findings demonstrate that HB is an insertion and deletion mutant of DuAdV-3.

Synthesis of Poly(thioester sulfonamide)s via the Ring-Opening Copolymerization of Cyclic Thioanhydride with N-Sulfonyl Aziridine Using Mild Phosphazene Base.

Providing access to diverse polymer structures is highly desirable, which helps to explore new polymer materials. Poly(thioester sulfonamide)s, combining both the advantages of thioesters and amides, however, are rarely available in polymer chemistry. Here, the ring-opening copolymerization (ROCOP) of cyclic thioanhydride with N-sulfonyl aziridine using mild phosphazene base, resulting in well-defined poly(thioester sulfonamide)s with highly alternative structures, high yields, and controlled molecular weights, is reported. Additionally, benefiting from the mild catalytic process, this ROCOP can be combined with ROCOP of N-sulfonyl aziridines with cyclic anhydrides to produce novel block copolymers.

The effect of infertile semen on the mRNA-based body fluid identification.

In the past decade, mRNA markers have been well demonstrated as promising molecular markers in forensic body fluid identification (BFI), and successfully used in wide applications. Several studies have assessed the performance of semen-specific mRNA markers in distinguishing semen from other common body fluids at the crime scene. Infertility has been reported as a global health problem that is affecting approximately 15% of couples worldwide. Therefore, it is important for forensic researchers to consider the impact of infertility on semen identification. This study aimed to explore the effect of semen from infertile men (hereinafter "infertile semen") on BFI and to identify semen-specific mRNAs that can efficiently and accurately distinguish normal and infertile semen samples from other body fluids. Results showed that the selected five mRNAs (KLK3, TGM4, SEMG1, PRM1, and PRM2) performed a significantly high semen specificity in normal semen. Moreover, KLK3 was slightly influenced by infertile semen samples with over 98% positive results in all semen samples. The accuracy to predict normal semen reached up to 96.6% using the discrimination function Y1 with KLK3 and PRM1. However, when the infertile semen samples were included in discrimination function (function Y2 with KLK3), the accuracy rate of semen identification (including the normal and infertile semen) was down to 89.5%. Besides, the sensitivity of multiplex assay could reach down to 50pg. Our results suggest that it is important to consider the presence of infertile semen when using mRNAs to identify semen samples, which would have a far-reaching impact in forensic identification.

Feasibility of using probabilistic methods to analyse microRNA quantitative data in forensically relevant body fluids: a proof-of-principle study.

Several studies have confirmed that microRNAs (miRNAs) are promising markers for body fluid identification since they were introduced to this field. However, there is no consensus on the choice of reference genes and identification strategies. In this study, 13 potential candidate miRNAs were screened from three forensically relevant body fluid datasets, and the expression of 12 markers in five body fluids was determined using a real-time quantitative method. Two probabilistic approaches, Naive Bayes (NB) and partial least squares discriminant analysis (PLS-DA), were then applied to predict the origin of the samples to determine whether probabilistic methods are helpful in body fluid identification using miRNA quantitative data. Furthermore, 14 reference combinations were used to validate the influence of different reference choices on the predicted results simultaneously. Our results showed that in the NB model, leave-one-out cross-validation (LOOCV) achieved 100% accuracy and the prediction accuracy of the test set was 100% in most reference combinations. In the PLS-DA model, the first two components could interpret about 80% expression variance and LOOCV achieved 100% accuracy when miR-92a-3p was used as the reference. This study preliminarily proved that probabilistic approaches hold huge potential in miRNA-based body fluid identification, and the choice of references influences the prediction results to a certain extent.

Establishment and evaluation of an indirect ELISA for detection of antibodies to goat Klebsiella pneumonia.

BACKGROUND: Klebsiella pneumonia, a Gram-negative bacterium belonging to the genus Enterobacter, causes many human and livestock diseases. Notably, infected goats may develop pneumonia, septicemia, which can lead to occasional death, resulting in great economic losses in goat-farming industry. However, there are little systematic methods for detection of goat Klebsiella pneumoniae in livestock production. RESULTS: In this study, we developed a Klebsiella pneumoniae goat polyclonal antibody and established an indirect ELISA method to detect the Klebsiella pneumoniae. After screening and optimizing the conditions for detection, we determined the optimal working dilutions of the coated-bacterial antigen, the polyclonal antibody, and the enzyme-labeled secondary antibody that were 1:800 (2.99 × 107 CFU/ml), 1:6400, and 1:5000, respectively. The optimal condition of coating and blocking were both 4 °C for 12 h. The optimal dilution buffers of bacterial antigen, the antibodies, and the blocking buffer were 0.05 mol/L carbonate buffer, 1% BSA phosphate buffer, and 1.5% BSA carbonate buffer, respectively. The cut-off value was determined to be 0.28, and the analytical sensitivity was 1:800 (dilution of a positive sample). Furthermore, there was no cross-reaction between the coated antigen and goat serum positive for antibodies against other bacteria, indicating that indirect ELISA could detect Klebsiella pneumoniae specifically in most cases. The average coefficients of variation of intra-assay and inter-assay were 4.37 and 5.17% indicating favorable reproducibility of indirect ELISA. In the detection of clinical veterinary samples, the positive rate of indirect ELISA was 6.74%, higher than that of conventional agglutination assays. CONCLUSIONS: Taken together, we successfully established an indirect ELISA method for detecting antibodies against Klebsiella pneumoniae in goats, which can be applied in production.

Postmortem interval determination using mRNA markers and DNA normalization.

Postmortem interval (PMI) determination is an important part of criminal investigations, but it is still subject to uncertainty. Degradation of mRNA in PMI determination has been studied in decays; however, some studies have reported no correlation between PMI and RNA degradation. Thus, we aimed to determine whether RNA quantity was correlated with PMI. Heart and brain tissues were separated from a mouse model of a 0-48 h PMI with 29 time points. We then coextracted the DNA and RNA in one tube with Bioteke coextraction kits and selected some mRNA markers associated with cell oxygen deprivation and apoptosis as target genes, such as hypoxia-associated factor (HAF), apoptosis-inducing factor (AIF), hypoxia-inducible factor 2 alpha (HIF2a), and factor inhibiting HIF (FIH). We measured the quantity of these markers using real-time quantitative PCR (qPCR), and Caspase-3 DNA and 18S were each used for normalization. The results showed that in the heart tissue, the degradation of HIF2a, AIF, and FIH was correlated with PMI, as was the degradation of HIF2a, FIH, and AIF in brain tissue when normalized with Caspase-3 DNA. However, when normalized with 18S, only the degradation of HIF2a in brain tissue was correlated with PMI. Interestingly, the quantity of HAF in brain tissue was found to increase after death with either 18S or Caspase-3 DNA normalization, and it was significantly correlated with 0-48 h PMI. These results indicated that mRNA quantity can be used to determine PMI and that Caspase-3 DNA is feasible for PMI estimation. In summary, we established mathematical models for PMI determination using multiple mRNA markers and multiple tissues and further studies are needed to validate and investigate these markers and mathematical models in human tissues.Duo Peng and Meili Lv contributed equally to this work.

Correction to: Electroacupuncture Upregulated Ghrelin in Rats with Functional Dyspepsia via AMPK/TSC2/Rheb­Mediated mTOR Inhibition.

The original version of the article is missing the grant information in Acknowledgments section. Corrected version of Acknowledgments is given below.

Electroacupuncture Upregulated Ghrelin in Rats with Functional Dyspepsia via AMPK/TSC2/Rheb-Mediated mTOR Inhibition.

BACKGROUND: Gastrointestinal motility disorder is an important pathological basis for functional dyspepsia (FD). Epigastric ache and discomfort are the main symptoms of FD, and ghrelin deficiency is closely related to the occurrence and development of FD. While electroacupuncture (EA) alleviated the symptoms of FD patients and improved their quality of life, there is a lack of sufficient mechanistic evidence to support these beneficial effects. METHODS: An in vivo FD model was established in wild-type and mammalian target of rapamycin (mTOR) knockout (-/-) rats. FD rats were subjected to EA with or without mTOR agonists or inhibitors. Gastric emptying and intestinal propulsion were assessed, and pathological changes in the hypothalamus, gastric antrum, and small intestine were examined histologically. In addition, ghrelin expression and AMPK/TSC2/Rheb/mTOR activation were detected by quantitative reverse transcription polymerase chain reaction and western blot. RESULTS: EA alone or in combination with mTOR inhibitors improved gastrointestinal function in FD rats by increasing the rates of intestinal propulsion and gastric emptying, and pathological changes in the hypothalamus, gastric antrum, and small intestine were alleviated. This may be related to the significant upregulation of ghrelin expression and the effective activation of the AMPK/TSC2/Rheb/mTOR signaling pathway. Interestingly, EA also improved gastrointestinal function and ghrelin expression in mTOR (-/-) KO FD rats. CONCLUSION: Altering the level of ghrelin by regulating AMPK/TSC2/Rheb-mediated mTOR inhibition is an important way through which EA treats FD. The complex EA-mediated regulatory mechanisms of the brain-gut axis still require further exploration.

Genetic polymorphisms of 17 Y-chromosomal STRs in the Chengdu Han population of China.

Chengdu is located at the center of Sichuan Province in southwestern China, and its primary demographic group is the Han population. The aim of this study was to contribute data detailing 17 Y-short tandem repeat (Y-STR) loci from 3291 Chengdu Han male samples analyzed with the AmpFLSTR® Yfiler® PCR Amplification Kit. We observed 2228 different haplotypes, and haplotype diversity (HD) was 0.9992. Gene diversity (GD) values for the 17 Y-STR loci of the Chengdu Han population ranged from 0.4156 to 0.9529. Haplotype match probability (HMC) was 0.0011. Compared with 13 reference populations of six provinces surrounding Chengdu, we observed that the Chengdu Han population was significantly different from each of these populations.

Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi.

BACKGROUND: The protozoan parasite Trypanosoma cruzi, causative agent of Chagas disease, depends upon a cell surface-expressed trans-sialidase (ts) to avoid activation of complement-mediated lysis and to enhance intracellular invasion. However these functions alone fail to account for the size of this gene family in T. cruzi, especially considering that most of these genes encode proteins lacking ts enzyme activity. Previous whole genome sequencing of the CL Brener clone of T. cruzi identified ~1400 ts variants, but left many partially assembled sequences unannotated. RESULTS: In the current study we reevaluated the trans-sialidase-like sequences in this reference strain, identifying an additional 1779 full-length and partial ts genes with their important features annotated, and confirming the expression of previously annotated "pseudogenes" and newly annotated ts family members. Multiple EM for Motif Elicitation (MEME) analysis allowed us to generate a model T. cruzi ts (TcTS) based upon the most conserved motif patterns and demonstrated that a common motif order is highly conserved among ts family members. Using a newly developed pipeline for the analysis of recombination within large gene families, we further demonstrate that TcTS family members are undergoing frequent recombination, generating new variants from the thousands of functional and non-functional ts gene segments but retaining the overall structure of the core TcTS family members. CONCLUSIONS: The number and variety as well as high recombination frequency of TcTS family members supports strong evolutionary pressure, probably exerted by immune selection, for continued variation in ts sequences in T. cruzi, and thus for a unique immune evasion mechanism for the large ts gene family.

Genome-wide analysis reveals origin of transfer RNA genes from tRNA halves.

Transfer RNAs (tRNAs) play an important role linking mitochondrial RNA and amino acids during protein biogenesis. Four types of tRNA genes have been identified in living organisms. However, the evolutionary origin of tRNAs remains largely unknown. In this article, we conduct a deep sequence analysis of diverse genomes that cover all three domains of life to unveil the evolutionary history of tRNA genes from tRNA halves. tRNA half homologs were detected in diverse organisms, and some of them were expressed in mouse tissues. Continuous tRNA genes have a conserved pattern similar to indels, which is, more closely flanking regions have higher single nucleotide substitution rates, whereas tRNA half homologs do not have this pattern. In addition, tRNAs tend to break into tRNA halves when tissues are incubated in vitro, the tendency of tRNA to break into tRNA halves may be a "side-effect" of tRNA genes evolving from tRNA halves. These results suggest that modern tRNAs originated from tRNA halves through a repeat element-mediated mechanism. These findings provide insight into the evolutionary origin of tRNA genes.

Iron, Copper, and Zinc Homeostasis in the Battle between Macrophage and Mycobacterium Tuberculosis.

Iron, copper, and zinc play integral roles in the battle against Mycobacterium tuberculosis (Mtb) infection; however, they are often trapped between nutrients and toxins, posing a significant challenge to macrophages and Mtb to utilize them. Due to this two-sided effect, macrophages and Mtb strictly regulate metal uptake, storage, and excretion. This review discusses the balanced regulation of iron, copper, and zinc in macrophages and Mtb during infection, focusing on the intracellular metal regulatory system. Macrophages typically use the two-sided effect of metals to limit Mtb access to nutrients or poison them. Mtb has developed a metal metabolism regulatory mechanism compatible with the nutritional immune strategy. This includes the mediation of relevant metalloproteins and metalloenzymes to maintain the multimetal balance. This review also explored the regulation of metal metabolism homeostasis in macrophages resistant to Mtb infection, providing a theoretical foundation for identifying potential clinical targets for Mtb infection, developing metalloid anti-tuberculosis drugs, and understanding the immune mechanisms against intracellular Mtb infection.

Brain network functional connectivity changes in long illness duration chronic schizophrenia.

Introduction: Chronic schizophrenia has a course of 5 years or more and has a widespread abnormalities in brain functional connectivity. This study aimed to find characteristic functional and structural changes in a long illness duration chronic schizophrenia (10 years or more). Methods: Thirty-six patients with a long illness duration chronic schizophrenia and 38 healthy controls were analyzed by independent component analysis of brain network functional connectivity. Correlation analysis with clinical duration was performed on six resting state networks: auditory network, default mode network, dorsal attention network, fronto-parietal network, somatomotor network, and visual network. Results: The differences in the resting state network between the two groups revealed that patients exhibited enhanced inter-network connections between default mode network and multiple brain networks, while the inter-network connections between somatomotor network, default mode network and visual network were reduced. In patients, functional connectivity of Cuneus_L was negatively correlated with illness duration. Furthermore, receiver operating characteristic curve of functional connectivity showed that changes in Thalamus_L, Rectus_L, Frontal_Mid_R, and Cerebelum_9_L may indicate a longer illness duration chronic schizophrenia. Discussion: In our study, we also confirmed that the course of disease is significantly associated with specific brain regions, and the changes in specific brain regions may indicate that chronic schizophrenia has a course of 10 years or more.