Comprehensive molecular evolutionary analysis of small heat shock proteins in five diploid Gossypium species.

The small heat shock proteins (sHSPs) are important components in plant growth and development, and stress response. However, a systematical understanding of the sHSP family is yet to be reported in five diploid Gossypium species. In this study, 34 GlsHSPs, 36 GrsHSPs, 37 GtsHSPs, 37 GasHSPs, and 38 GhesHSPs were identified in Gossypium longicalyx, Gossypium raimondii, Gossypium turneri, Gossypium arboreum, and Gossypium herbaceum, respectively. These sHSP members can be clustered into 10 subfamilies. Different subfamilies had different member numbers, motif distributions, gene structures, gene duplication events, gene loss numbers, and cis-regulatory elements. Besides, the paleohexaploidization event in cotton ancestor led to expanding the sHSP members and it was also inherited by five diploid Gossypium species. After the cotton ancestor divergence, the sHSP members had the relatively conserved evolution in five diploid Gossypium species. The comprehensive evolutionary history of the sHSP family was revealed in five diploid Gossypium species. Furthermore, several GasHSPs and GhesHSPs were important candidates in plant growth and development, and stress response. These current findings can provide valuable information for the molecular evolution and further functional research of the sHSP family in cotton.

A comparison of the determination of multiple pesticide residues in fruits, vegetables, and edible fungi using gas chromatography combined with filtration purification and solid-phase extraction.

The multiplug filtration clean-up (m-PFC) and solid-phase extraction (SPE) pretreatment methods were employed to process 8 representative matrices in fruits, vegetables, and edible fungi, respectively. 37 pesticide residues were determined using gas chromatography equipped with ECD and FPD detectors. The measurement data were compared and analyzed following m-PFC purification and gas chromatography analysis, and both accuracy and precision met the (EU) 2021/808 requirements, achieving recovery rates for the 8 matrices ranging from 67.0% to 112.8% (averaging over 83.8% recovery), and RSDs between 0.2% and 15.2%. The 37 pesticides exhibited good linearity between 0.05 and 1.6 µg mL-1, and the matrix effect was found to be weaker compared to that of the Florisil solid-phase extraction method. The detection limits ranged from 0.0001 to 0.03 µg kg-1, with 31 pesticides showing lower detection limits compared to the SPE method. The application of this method to 150 real samples resulted in the detection of 17 pesticides across all samples. Fewer pigments were detected in m-PFC purified solutions compared to Florisil PR SPE when analyzed by liquid chromatography. m-PFC achieved more thorough adsorption of endogenous substances like pigments, reducing instrument contamination, utilizing less organic solvent, and simplifying the operation. This purification step offers clear advantages, allowing for the processing of larger sample batches in a short time. It can serve as a replacement for SPE methods like Florisi PR in batch detection of fruit and vegetable samples.

Treg Immunomodulation Contributes to the Anti-atherosclerotic Effects of Huxin Formula in ApoE-/- Mice.

OBJECTIVE: To explore the effects of Huxin formula (HXF) in curtailing atherosclerosis and its underlying mechanism. METHODS: According to random number table method, 24 specific pathogen free male ApoE-/- mice were randomly divided into model group, HXF low-dose (HXF-L) group (8.4 g/kg daily), HXF high-dose (HXF-H) group (16.8 g/kg daily), and pravastatin (8 mg/kg daily) group in Experiment I (n=6 per group). C57BL/6J mice served as the control group (n=6). ApoE-/- mice in HXF-L, HXF-H, pravastatin groups were fed a Western diet and administered continuously by gavage for 12 weeks, while C57BL/6J mice in the control group were fed conventional lab mouse chow for 12 weeks. Further, Tregs were depleted by weekly intraperitoneal injection of purified anti-mouse CD25 antibody (PC61, 250 µg per mouse) for 4 weeks in Experiment II (n=6 per group). Oil Red O and Masson staining were used to evaluate the plaque area and aortic root fibrosis. The CD4+CD25+Foxp3+Treg counts in the lymph nodes and spleen cells were detected using flow cytometric analysis. The transforming growth factor-ß1 (TGF-ß1), interleukin (IL)-10, and IL-6 serum levels were examined by MILLIPLEX® MAP technology. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot were utilized to assess the expression of TGF-ß mRNA and protein in the aorta. The expression of CD4+T lymphocytes, macrophages and smooth muscle cells in the aortic root were detected by immunofluorescence staining. RESULTS: HXF reduced plaque area in ApoE-/- mice (P<0.01). HXF increased the Treg counts in the lymph nodes and spleen cells (P<0.05 or P<0.01). Moreover, HXF alleviated inflammatory response via elevating IL-10 and TGF-ß 1 serum levels (P<0.05), while decreasing the IL-6 serum levels in ApoE-/- mice (P>0.05). Also, HXF upregulated the expression of TGF-ß mRNA and protein in the aorta (P<0.05). Additionally, HXF attenuated CD4+T lymphocytes, macrophages and smooth muscle cells in aortic root plaque (P<0.01). Furthermore, the depletion of Tregs with CD25 antibody (PC61) curtailed the reduction in plaque area and aortic root fibrosis by HXF (P<0.01). CONCLUSION: HXF relieved atherosclerosis, probably by restraining inflammatory response, reducing inflammatory cell infiltration and attenuating aortic root fibrosis by increasing Treg counts.

Enantiospecific Analysis of Carboxylic Acids Using Cinchona Alkaloid Dimers as Chiral Solvating Agents.

Cinchona alkaloid derivatives as Brønsted base catalysts have attracted considerable attention in the field of asymmetric catalysis. However, their potential application as chiral solvating agents has not been described. In this research, we investigated the use of the Cinchona alkaloid dimer, namely, (DHQ)2PHAL, as a chiral solvating agent for discerning various mandelic acid derivatives through 1H NMR spectroscopy. The addition of catalytic amounts of DMAP facilitated this process. Our experimental results demonstrate that dimeric (DHQ)2PHAL exhibits remarkable chiral discrimination properties regarding the diagnostic split protons of 1H NMR signals (including 24 examples, up to 0.321 ppm). Furthermore, it serves as an excellent chiral discriminating agent and provides good resolution for racemic chiral phosphoric acid as determined by 31P NMR spectroscopy. The quality of enantiodifferentiation has also been evaluated by means of the parameter "resolution (Rs)". Significantly, this class of CSAs based on (alkaloid)2linker systems with an azaaromatic linker can be directly employed, which is commercially available in an enantiopure form at very low cost and exhibits promising potential in determining the enantiopurity of α-hydroxy acids by chemoselective and biocatalytic reactions.

Development of Pure Certified Reference Material of Cannabidiol.

Cannabidiol (CBD) is the major functional component in hemp and has a broad range of pharmacological applications, such as analgesic, anti-epileptic, anti-anxiety, etc. Currently, CBD is widely used in pharmaceuticals, cosmetics, and food. To ensure the quality and safety of the products containing CBD, more and more related sample testing is being conducted, and the demand for CBD-certified reference material (CRM) has also sharply increased. However, there is currently a lack of relevant reference materials. In this paper, a simple method for preparing CBD CRM was established based on preparative liquid chromatography using crude hemp extract as a raw material. A qualitative analysis of CBD was performed using techniques such as ultraviolet absorption spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR), and differential scanning calorimetry (DSC). High-performance liquid chromatography (HPLC) was used for the homogeneity and stability tests, and the data were analyzed using an F-test and a T-test, respectively. Then, eight qualified laboratories were chosen for the determination of a certified value using HPLC. The results show that the CBD CRM had excellent homogeneity and good stability for 18 months. The certified value was 99.57%, with an expanded uncertainty of 0.24% (p = 0.95, k = 2). The developed CBD CRM can be used for the detection and quality control of cannabidiol products.

Nondestructive identification and classification of starch types based on multispectral techniques coupled with chemometrics.

Starch is the main source of energy and nutrition. Therefore, some merchants often illegally add cheaper starches to other types of starches or package cheaper starches as higher priced starches to raise the price. In this study, 159 samples of commercially available wheat starch, potato starch, corn starch and sweet potato starch were selected for the identification and classification based on multispectral techniques, including near-infrared (NIR), mid-infrared (MIR) and Raman spectroscopy combined with chemometrics, including pretreatment methods, characteristic wavelength selection methods and classification algorithms. The results indicate that all three spectral techniques can be used to discriminate starch types. The Raman spectroscopy demonstrated superior performance compared to that of NIR and MIR spectroscopy. The accuracy of the models after characteristic wavelength selection is generally superior to that of the full spectrum, and two-dimensional correlation spectroscopy (2D-COS) achieves better model performance than other wavelength selection methods. Among the four classification methods, convolutional neural network (CNN) exhibited the best prediction performance, achieving accuracies of 99.74 %, 97.57 % and 98.65 % in NIR, MIR and Raman spectra, respectively, without pretreatment or characteristic wavelength selection.

Comparative key aroma compounds and sensory correlations of aromatic coconut water varieties: Insights from GC × GC-O-TOF-MS, E-nose, and sensory analysis.

Aroma is a key criterion in evaluating aromatic coconut water. A comparison regarding key aroma compounds and sensory correlations was made between Thailand Aromatic Green Dwarf (THD) and Cocos nucifera L. cv. Wenye No. 4 coconut water using E-nose and GC × GC-O-TOF-MS combined with chemometrics. Twenty-one volatile components of coconut water were identified by GC × GC-O-TOF-MS, and 5 key aroma compounds were analyzed by relative odor activity value and aroma extract dilution analysis. Moreover, the combination of the E-nose with orthogonal partial least squares was highly effective in discriminating between the two coconut water samples and screened the key sensors responsible for this differentiation. Additionally, the correlation between volatile compounds and sensory properties was established using partial least squares. The key aroma compounds of coconut water exhibited positive correlations with the corresponding sensory properties.

Nomogram based on CMTM6 expression and clinical characteristics to predict postoperative overall survival in patients with hepatocellular carcinoma.

BACKGROUND: The purpose of this study was to investigate the expression of CMTM6 in HCC tissues and its prognostic value, and to try to develop a nomogram prognostic model based on CMTM6. METHODS: In this retrospective study, immunohistochemical (IHC) staining was performed in 178 patients who underwent radical hepatectomy in the same surgical team. R software was used to construct the nomogram model. The Bootstrap sampling method was used for internal validation. RESULTS: CMTM6 is significantly expressed in HCC tissues and is closely associated with decreased overall survival (OS). PVTT (HR = 6.2, 95% CI: 3.06 12.6, P<0.001), CMTM6 (HR=2.30, 95% CI: 1.27 4.0, P=0.006) and MVI (HR=10.8, 95% CI: 4.19-27.6, P<0.001) were independent predictors of OS. The nomogram combined with CMTM6, PVTT and MVI was more predictive than the traditional TNM scoring system, and the prediction effects of 1-year and 3-year OS were accurate. CONCLUSIONS: The prognosis of a patient may be predicted using high levels of CMTM6 expression in HCC tissues, and the nomogram model including CMTM6 expression has the best predictive ability.

Polyarylisocyanides Derived from an Alkyne-Pd(II) Catalyst as Robust Alignment Media with Excellent Enantiodiscimination.

The development of chiral alignment media for measuring anisotropic NMR parameters provides an opportunity to determine the absolute configuration of chiral molecules without the need for derivatization. However, chiral alignment media with a high and robust enantiodiscriminating property for a wide range of chiral molecules are still scarce. In this study, we synthesized cholesterol-end-functionalized helical polyisocyanides from a chiral monomer using a cholesterol-based alkyne-Pd(II) initiator. These stereoregular polyisocyanides form stable and weak anisotropic lyotropic liquid crystals (LLCs) in dichloromethane systems, exhibiting highly optical activities in both single left- and right-handed helices. The preparation process of the media was straightforward, and the aligning property of the LLCs could be controlled by adjusting the concentration and temperature. Using the chiral polyisocyanides, we extracted the residual dipolar coupling for an enantiomeric pair of isopinocampheol (IPC), as well as a number of pharmaceutical molecules, demonstrating excellent enantiodiscriminating properties for a broad range of chiral compounds.

Health system barriers to timely routine measles vaccinations in rural southwest China: a qualitative study on the perspectives of township vaccination professionals and village doctors.

OBJECTIVES: A well-functioning health system ensures timely routine measles vaccinations for age-appropriate children, minimising measles risk. However, there is limited knowledge about the impact of the performance of immunisation programmes in health systems on the timeliness of measles vaccination. This study aimed to identify health system barriers to timely routine measles vaccination in rural southwest China, integrating the perspectives of township vaccination professionals and village doctors. DESIGN, SETTING AND PARTICIPANTS: Qualitative study among township vaccination professionals and village doctors in rural Guangxi, southwest China. METHODS: 20 focus group discussions (FGDs) at township level and 120 in-depth interviews (IDIs) at village level, based on a four-theme framework. We used convenience sampling to recruit 60 township vaccination professionals and 120 village doctors in 2015. Instruments used were a semistructured questionnaire and interview outlines. We collected township and village-level data focusing on themes of health resources allocation, pattern of vaccination services, management and supervision of vaccination services, and perceptions of vaccination policy. The FGDs and IDIs were audio-recorded and transcribed. Braun and Clarke's thematic analysis approach was adopted to synthesise findings into meaningful subthemes, narrative text and illustrative quotations. RESULTS: The health system barriers to timely routine vaccinations were explored across four themes. Barriers in the health resources allocation theme comprised (1) inadequacy of vaccination-related human resources (eg, lack of township vaccination professionals and lack of young village doctors), and (2) incompatible and non-identical information system of vaccination services across regions. Barriers in the pattern of vaccination services theme included inflexible vaccination services models, for example, routine vaccination services being offered monthly on fixed vaccination days, limited numbers of vaccination days per month, vaccination days being set on non-local market days, vaccination days being clustered into a specific period and absence of formal vaccination appointments. Ineffective economic incentive mechanism was identified as a barrier in the management and supervision of vaccination services theme. Low-degree participation of village doctors in routine vaccination services was identified as a barrier in the perceptions of vaccination policy theme. CONCLUSIONS: We encourage policymakers and stakeholders to apply these findings to improve the timeliness of routine vaccination. Barriers to timely routine vaccination include inadequate allocation of vaccination-related resources and inflexible vaccination service delivery models. Financial and non-financial incentives should be used to retain and recruit vaccination professionals and village doctors. Strengthening information systems with unified data standards enables cross-regional data exchange. Optimising immunisation services and rationalising vaccination days could eliminate health system barriers and improve vaccination timeliness in rural China.

Integration of transcriptome and metabolome analyses reveals the role of OsSPL10 in rice defense against brown planthopper.

KEY MESSAGE: OsSPL10 is a negative regulator of rice defense against BPH, knockout of OsSPL10 enhances BPH resistance through upregulation of defense-related genes and accumulation of secondary metabolites. Rice (Oryza sativa L.), one of the most important staple foods worldwide, is frequently attacked by various herbivores, including brown planthopper (BPH, Nilaparvata lugens). BPH is a typical monophagous, phloem-sucking herbivore that has been a substantial threat to rice production and global food security. Understanding the regulatory mechanism of defense responses to BPH is essential for improving BPH resistance in rice. In this study, a SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE 10 (OsSPL10) transcription factor was found to play a negative role in the defenses of rice against BPH. To gain insights into the molecular and biochemical mechanisms of OsSPL10, we performed combined analyses of transcriptome and metabolome, and revealed that knockout of OsSPL10 gene improved rice resistance against BPH by enhancing the direct and indirect defenses. Genes involved in plant hormone signal transduction, MAPK signaling pathway, phenylpropanoid biosynthesis, and plant-pathogen interaction pathway were significantly upregulated in spl10 mutant. Moreover, spl10 mutant exhibited increased accumulation of defense-related secondary metabolites in the phenylpropanoid and terpenoid pathways. Our findings reveal a novel role for OsSPL10 gene in regulating the rice defense responses, which can be used as a potential target for genetic improvement of BPH resistance in rice.

HSC-derived exosomal miR-199a-5p promotes HSC activation and hepatocyte EMT via targeting SIRT1 in hepatic fibrosis.

Exosomes have been implicated in inflammation-related diseases, such as hepatic fibrosis (HF) and renal fibrosis, via transferring bioactive cargoes to recipient cells. This study aimed to investigate the possible effect of hepatic stellate cell (HSC)-derived exosomes on the initiation and development of HF by delivering microRNA (miR)-199a-5p. In HF rats with cholestasis induced by ligating the common bile duct, miR-199a-5p was upregulated while SIRT1 was downregulated in liver tissues from bile duct ligation (BDL) rats compared with that of sham rats. Furthermore, miR-199a-5p expression was upregulated, but the mRNA and protein expression levels of SIRT1 were downregulated in TGF-ß1-activated LX-2. miR-199a-5p promoted the proliferation and further activation of LX-2 and enhanced the expression levels of the HF markers COL1A1 and α-SMA. Subsequently, the binding of miR-199a-5p to the 3'UTR of SIRT1 mRNA was predicted by bioinformatics websites and evidenced by fluorescent reporter assay. Knocking down SIRT1 enhanced the abilities of LX-2 cell proliferation, migration, and colony formation and increased the expression levels of the HF markers α-SMA and COL1A1. LX-2-derived exosomal miR-199a-5p transferred to LX-2 and THLE-2, inhibited the proliferation of THLE-2, and promoted the epithelial mesenchymal transition (EMT) and senescence of THLE-2. Furthermore, in vivo results suggested that miR-199a-5p overexpression aggravated HF in BDL rats; increased miR-199a-5p, α-SMA, and COL1A1 expression levels; and significantly upregulated the serum ALT, AST, TBA, and TBIL levels. However, reverse results were obtained with inhibited miR-199a-5p expression. In conclusion, HSC-derived exosomal miR-199a-5p may promote HF by accelerating HSC activation and hepatocyte EMT by targeting SIRT1, suggesting that miR-199a-5p and SIRT1 may serve as potential therapeutic targets for HF.

[Effects of Polystyrene Microplastics on Growth, Physiology, Biochemistry, and Canopy Temperature Characteristics of Chinese Cabbage Pakchoi (Brassica chinensis L.)].

The aim of this study was to clarify the response characteristics of Chinese cabbage pakchoi (Brassica chinensis L.) under two particle size (100 nm and 1000 nm) polystyrene microplastic (PS-MPs) stress conditions. This study can provide a theoretical basis and experimental reference for the interpretation of the physiological and ecological mechanism of microplastic pollution and the bioremediation of microplastic-contaminated soil. Hydroponic experiments were carried out to study the effects of two particle sizes (100 nm and 1000 nm) of PS-MPs on growth, photosynthetic physiology, antioxidant enzyme activities, nutritional quality, anatomical structure, and canopy temperature in Chinese cabbage pakchoi. The results showed that PS-MPs stress significantly inhibited the growth and development of Chinese cabbage pakchoi. When PS-MPs stress was increased, the phenotypic indicators were significantly reduced. Meanwhile, PS-MPs stress significantly enhanced the oxidative stress response of Chinese cabbage pakchoi, such as the activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX) and the content of malondialdehyde (MDA) in leaves. Such a change tended to decrease the thickness of fenestrated and leaf and spongy tissues. Moreover, PS-MPs stress significantly increased the canopy population temperature of the Chinese cabbage pakchoi leaves. Microplastic stress had obvious inhibitory effects and toxic damage on the growth, development, and physical and chemical properties of Chinese cabbage pakchoi.

Self-reported critical gaps in the essential knowledge and capacity of spatial epidemiology between the current university education and competency-oriented professional demands in preparing for a future pandemic among public health postgraduates in China: a nationwide cross-sectional survey.

BACKGROUND: Spatial epidemiology plays an important role in public health. Yet, it is unclear whether the current university education in spatial epidemiology in China could meet the competency-oriented professional demands. This study aimed to understand the current situation of education and training, practical application, and potential demands in spatial epidemiology among public health postgraduates in China, and to assess the critical gaps in a future emerging infectious diseases (EID) pandemic preparedness and response. METHODS: This study was divided into three parts. The first part was a comparative study on spatial epidemiology education in international public health postgraduate training. The second part was a cross-sectional survey conducted among public health professionals. The third part was a nationwide cross-sectional survey conducted among public health postgraduates at Chinese universities from October 2020 to February 2021. Data was collected by the WeChat-based questionnaire star survey system and analyzed using the SPSS software. RESULTS: International education institutions had required public health postgraduates to master the essential knowledge and capacity of spatial epidemiology. A total of 198 public health professionals were surveyed, and they had a median of 4.00 (IQR 3.13-4.53) in demand degree of spatial epidemiology. A total of 1354 public health postgraduates were surveyed from 51 universities. Only 29.41% (15/51) of universities offered spatial epidemiology course. Around 8.05% (109/1354) of postgraduates had learned spatial epidemiology, and had a median of 1.05 (IQR 1.00-1.29) in learning degree and a median of 1.91 (IQR 1.05-2.78) in practical application degree of spatial epidemiology. To enhance professional capacity, 65.95% (893/1354) of postgraduates hoped that universities would deliver a credit-course of spatial epidemiology. CONCLUSIONS: A huge unmet education and training demand in spatial epidemiology existed in the current education system of public health postgraduates in China. To enhance the competency-oriented professional capacity in preparedness and response to a future pandemic, it is urgent to incorporate the teaching and training of spatial epidemiology into the compulsory curriculum system of public health postgraduates in China.

Two and three-dimensional halogen-bonded frameworks: self-assembly influenced by crystallization solvents.

In this paper, two types of solid phase 2D and 3D XBOFs were selectively constructed from identical building blocks of tetraphenylmethane tetrapyridine derivative and 1,4-diiodotetrafluorobenzene by changing the crystallization solvent. This 3D XBOF is a novel hybrid supramolecular organic framework with the synergistic control of hydrogen and halogen bonds.

Fabrication of sodium alginate-doped carbon dot composite hydrogel and its application for La (III) adsorption and enhanced the removal of phosphorus.

Adsorption is an effective method for the removal of hazardous substances from wastewater. In this work, a low-cost and environmental-friendly composite hydrogel material of sodium alginate doped with nitrogen doped carbon dots (SA@NCDs) was fabricated by impregnation for lanthanide and enhanced phosphorus adsorption in wastewater. The effects of NCDs doping amount, dosage, pH, initial solution concentration, adsorption time and temperature on the process of La (III) adsorption by SA@NCDs were investigated. The adsorption isotherms fitted to Langmuir isotherm model (R2 = 0.9970-0.9989) and the adsorption kinetics followed pseudo-second-order kinetic model (R2 = 0.9992). The maximum adsorption capacity of the adsorbent for La (III) was 217.39 mg/g according to the Langmuir model at 298.15 K. After five cycles, the removal efficiency of La (III) adsorbed by SA@NCDs was still 85.1%. Moreover, the loaded La (III) enhanced the adsorption of phosphorus. The La (III)-SA@NCDs-5 hydrogel adsorbent greatly improved the adsorption capacity for phosphorus compared with the La (III)-free adsorbent, and the adsorption amount can reach 9.64 mg-P/g. The SA@NCDs complex hydrogels for rare earth adsorption were prepared by introducing NCDs rich in amino group into SA hydrogels. The introduction of NCDs increases the adsorption sites of hydrogels, and also overcomes the problem that NCDs itself is difficult to recover in wastewater treatment applications. The lanthanide adsorbed material has a stable structure and can be used to remove phosphorus to deal with waste using the waste. It indicates the SA@NCDs hydrogel composite adsorbent have good potential for wastewater treatment.

Research progress of applications for nano-materials in improved QuEChERS method.

The quick, easy, cheap, effective, rugged, and safe (QuEChERS) approach is widely used in sample pretreatment in agricultural products, food, environment, etc. And nano-materials are widely used in QuEChERS method due to its small size and large specific surface area. In this review, we examine the typical applications of several commonly used nano-materials in improved QuEChERS method. These materials include multi-walled carbon nanotubes (MWCNTs) and their derivatives, magnetic nanoparticles (MNPs), metal organic frameworks (MOFs), covalent organic frameworks (COFs), graphene oxide (GO), lipid and protein adsorbent (LPAS), cucurbituril (CBs), and carbon nano-cages (CNCs), and so on. The strengths and weaknesses of each nano-material are presented, as well as the challenging aspects that need to be addressed in future research. By comparing the applications and the current technology development, this review suggests utilizing artificial intelligence (AI) to screen suitable combinations of purification agents and performing virtual simulation experiments to verify the reliability of this methodology. By doing so, we aim to accelerate the development of new products and decrease the cost of innovation. It also recommends designing smarter pretreatment instruments to enhance the convenience and automation of the sample pretreatment process and reduce the margin for human error.

The PIWI-specific insertion module helps load longer piRNAs for translational activation essential for male fertility.

PIWI-clade proteins harness piRNAs of 24-33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR super-complex for translational activation. Importantly, we identify a c.1108C>T (p.R370W) mutation of HIWI (human PIWIL1) in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility.