A New Lysosome-Targeted NIR Fluorescent Probe for Specific Detection of Cysteine over Homocysteine and Glutathione.

In this paper, by modifying the thioxanthene-benzothiozolium fluorophore, BCy-Cys, a lysosome-targeted near-infrared (NIR) fluorescent probe was synthesized for the detection of cysteine (Cys) from homocysteine (Hcy)/glutathione (GSH). As expected, BCy-Cys exhibited high selectivity and high sensitivity for detection of Cys over Hcy/GSH, with an extremely low limit of detection at 0.31 µM, marked by obvious color changes. HRMS was conducted to confirm that the fluorescence intensity at 795 nm was significantly enhanced by the enhancement of intramolecular charge transfer (ICT). Importantly, BCy-Cys could be used to visualize both exogenous and endogenous lysosomal Cys, signifying its potential application in complex organismal systems.

CircKDM5B sponges miR-128 to regulate porcine blastocyst development by modulating trophectoderm barrier function.

Circular RNAs (circRNAs), which exert critical functions in the regulation of transcriptional and post-transcriptional gene expression, are found in mammalian cells but their functions in mammalian preimplantation embryo development remain poorly understood. Here, we showed that circKDM5B mediated miRNA-128 (miR-128) to regulate porcine early embryo development. We screened circRNAs potentially expressed in porcine embryos through an integrated analysis of sequencing data from mouse and human embryos, as well as porcine oocytes. An authentic circRNA originating from histone demethylase KDM5B (referred to as circKDM5B) was abundantly expressed in porcine embryos. Functional studies revealed that circKDM5B knockdown not only significantly reduced blastocyst formation but also decreased the number of total cells and trophectoderm (TE) cells. Moreover, the knockdown of circKDM5B resulted in the disturbance of tight junction assembly and impaired paracellular sealing within the TE epithelium. Mechanistically, miR-128 inhibitor injection could rescue the early development of circKDM5B knockdown embryos. Taken together, the findings revealed that circKDM5B functions as a miR-128 sponge, thereby facilitating early embryonic development in pigs through the modulation of gene expression linked to tight junction assembly.

Emamectin benzoate exposure impaired porcine oocyte maturation.

Emamectin benzoate (EB) is a widely used insecticide that can damage the central nervous and immune systems. EB exposure significantly reduced the number of eggs laid, hatching rate, and developmental rate of lower organisms such as nematodes. However, effects of EB exposure on the maturation of higher animals such as porcine oocytes remains unknown. Here we reported that EB exposure severely impaired porcine oocyte maturation. EB exposure with 200 µM prevented cumulus expansion and reduced the rates of first polar body (pb1) extrusion, cleavage and blastocyst after parthenogenetic activation. Moreover, EB exposure disrupted spindle organization, chromosome alignment, and polymerization of microfilaments, but also apparently decreased the levels of acetylated α-tubulin (Ac-Tub) in oocytes. In addition, EB exposure perturbed mitochondria distribution and increased levels of reactive oxygen species (ROS), but did not affect the distribution of cortical granules (CGs) in oocytes. Excessive ROS caused DNA damage accumulation and induced early apoptosis of oocytes. EB exposure led to the abnormal expression of cumulus expansion and apoptosis-associated genes. Altogether, these results demonstrate that EB exposure impaired nuclear and cytoplasmic maturation of porcine oocytes probably through oxidative stress and early apoptosis.

Inhibitory Effects of 3-Methylcholanthrene Exposure on Porcine Oocyte Maturation.

3-methylcholanthrene (3-MC) is a highly toxic environmental pollutant that impairs animal health. 3-MC exposure can cause abnormal spermatogenesis and ovarian dysfunction. However, the effects of 3-MC exposure on oocyte maturation and embryo development remain unclear. This study revealed the toxic effects of 3-MC exposure on oocyte maturation and embryo development. 3-MC with different concentrations of 0, 25, 50, and 100 µM was applied for in vitro maturation of porcine oocytes. The results showed that 100 µM 3-MC significantly inhibited cumulus expansion and the first polar body extrusion. The rates of cleavage and blastocyst of embryos derived from 3-MC-exposed oocytes were significantly lower than those in the control group. Additionally, the rates of spindle abnormalities and chromosomal misalignments were higher than those in the control group. Furthermore, 3-MC exposure not only decreased the levels of mitochondria, cortical granules (CGs), and acetylated α-Tubulin, but also increased the levels of reactive oxygen species (ROS), DNA damage, and apoptosis. The expression of cumulus expansion and apoptosis-related genes was abnormal in 3-MC-exposed oocytes. In conclusion, 3-MC exposure disrupted the nuclear and cytoplasmic maturation of porcine oocytes through oxidative stress.

Expression Profiling of Circular RNAs in Early Pregnant Jianghuai Buffaloes.

Circular RNA (circRNA) is expressed in cells and tissues of several species. However, the expression of circRNAs in the blood of Jianghuai buffaloes during early pregnancy has not been reported. In this study, we identified the DECs in the blood of Jianghuai buffaloes and annotated the functions of these DECs. The results showed that there were 890 DECs between the pregnant and non-pregnant groups, of which more than 80% were exon-derived circRNAs, including 323 up-regulated circRNAs and 567 down-regulated circRNAs. Enrichment analysis revealed that DECs were mainly enriched in the epidermal growth factor receptor-signaling pathway important for embryonic development and pregnancy maintenance. In addition, most DECs have multiple miRNA targets, suggesting that these DECs have the potential to function as miRNA sponges. In conclusion, several DECs are present between pregnant and non-pregnant Jianghuai buffaloes, and these DECs are associated with embryo implantation and pregnancy establishment.

A New Phenylazo-Based Fluorescent Probe for Sensitive Detection of Hypochlorous Acid in Aqueous Solution.

In this paper, we designed and synthesized a novel phenylazo-based fluorescent probe (RHN) for the sensing and imaging of hypochlorous acid (HClO) in mitochondria in living cells. In this process, HClO promoted the oxidation of the phenylazo group to generate a free Rhodol fluorophore moiety, which in turn restored strong fluorescence and realized the detection of HClO. As expected, RHN exhibited high selectivity, high sensitivity and rapid response, with detection limits as low as 22 nM (1.155 ng/mL). Importantly, the results of the cell imaging experiments indicated that RHN has the ability to image and sense HClO in mitochondria, which is of great significance for exploration of the specific role of HClO in both the immune system and diseases.

Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.

Rational design of a large Stokes shift xanthene-benzothiozolium dyad for probing cysteine in mitochondria.

Xanthene-modified cyanine dyes are considered to be an effective means to extend the emission wavelength and improve the photo-stability of cyanine dyes. However, the fluorophores obtained by this strategy generally have narrow Stokes shifts, which severely limits their application in biological imaging. Herein, a reasonable design strategy is adopted to provide an effective strategy to commendably improve the Stokes shift of xanthene-benzothiozolium fluorophores via the simultaneous expansion of a molecular π-conjugated system and heteroatomic substitution. Combined with density functional theory calculation guidance, the O atom is replaced with the S atom in the xanthene moiety and a π-conjugated benzene ring is introduced in the benzothiozolium moiety. Surprisingly, the results of the spectroscopic experiment showed that the fluorescence emission wavelength of PhCy-OH was extended to 803 nm, and the Stokes shift was improved to 68 nm. PhCy-Cys can effectively distinguish Cys from homocysteine (Hcy) and glutathione (GSH) with an extremely low detection limit of 0.166 µM. Importantly, PhCy-Cys has the ability to image endogenous Cys in mitochondria, providing the potential for exploring the specific function and mechanism of Cys in regulating oxidative stress in mitochondria.

A highly sensitive sensor for colorimetric detection of palladium(II) in lysosomes and its applications.

Considering the scarcity of palladium ion probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelength fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the toxicity of palladium ions at the subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe, BHCy-Pd, for the specific detection of palladium ions (Pd2+) in lysosomes via an internal charge-transfer (ICT) mechanism was designed and synthesized. As expected, BHCy-Pd exhibited a rapid, selective, and sensitive response for palladium with an ultralow limit of detection at 5.9 nM, accompanied by a distinct color change from purple to blue. Furthermore, BHCy-Pd can be made into a simple test strip for rapid and easy detection of Pd2+ in practical applications. Importantly, BHCy-Pd is capable of specific distribution in lysosomes, and thus can detect Pd2+ in real-time, thereby providing a potential tool for studying the cytotoxicity of Pd2+ ions at the subcellular level.

Circ_0000658 knockdown inhibits epithelial-mesenchymal transition in bladder cancer via miR-498-induced HMGA2 downregulation.

BACKGROUND: Epithelial-mesenchymal transition (EMT) has been associated with the angiogenesis and oncogenic phenotypes of multiple malignant tumors including bladder cancer (BCa). Circular RNAs (circRNAs) are recognized as crucial regulators in the EMT. This study aims to illustrate the possible role of circular RNA_0000658 (circ_0000658) in BCa and the underlying molecular mechanism. METHODS: The expression of circ_0000658, microRNA (miR)-498, and high mobility group AT-hook 2 (HMGA2) was assessed in cancer and adjacent normal tissue collected from BCa patients and human BCa cell lines (MGH-U3, T24, 5637 and SW780). BCa cells were transduced with a series of overexpression or shRNA plasmids to clarify the function of circ_0000658 and miR-498 on the oncogenic phenotypes and EMT of BCa cells. Further, we established nude mice xenografted with BCa cells to validate the roles of circ_0000658 on tumor growth in vivo. RESULTS: Circ_0000658 was highly expressed in BCa tissue samples and cell lines, which indicated a poor prognosis of BCa patients. Circ_0000658 competitively bound to miR-498 and thus restricted miR-498 expression. Meanwhile, circ_0000658 weakened the binding of miR-498 to the target gene HMGA2 and upregulated the HMGA2 expression. Circ_0000658 elevation or miR-498 knockdown augmented oncogenic phenotypes and EMT of BCa cells, corresponding to a reduction in the expression of ß-catenin and E-cadherin as well as an increase in the expression of N-cadherin, Slug, Snail, ZEB1 and Twist. Inhibition of HMGA2 reversed the effects of circ_0000658 overexpression on tumor growth in vivo. CONCLUSION: Altogether, our study uncovered the tumor-promoting role of circ_0000658 in BCa via the miR-498/HMGA2 axis.

A new near-infrared fluorescent probe for sensing extreme acidity and bioimaging in lysosome.

Since the intracellular pH plays an important role in the physiological and pathological processes, however, the probes that can be used for monitoring pH fluctuation under extreme acidic conditions are currently rare, so it is necessary to construct fluorescent probes for sensing pH less than 4. In this work, we developed a new near-infrared (NIR) fluorescent probeCy-SNNfor sensing pH fluctuation under extremely acidic conditions. For the preparation of this probe, benzothiozolium moiety was chosen as lysosomal targeting unit and NIR fluorophore, and barbituric acid moiety was fused in the polymethine chain of probe to introduce protonation center. Surprisingly, on the basis of the balance of quaternary ammonium salts and free amines, the pkavalue ofCy-SNNwas calculated as low as 2.96, implying thatCy-SNNcan be used in acidic conditions with pH < 4. Moreover,Cy-SNNexhibited highly selective response to H+over diverse analytes in real-time with dependable reversibility. Importantly,Cy-SNNcan be used to specifically target lysosome, providing potential tools for monitoring the function of lysosome in autophagy process.

Novel near-infrared spectroscopic probe for visualizing hydrogen sulfide in lysosomes.

Considering the scarcity of hydrogen sulfide (H2S) probes with subcellular organelle targeting, especially probes with near-infrared (NIR) emission wavelengths fluorophores, our group has been working to overcome this problem and looking forward to providing potential practical tools for exploring the relationship between the physiology and pathology of H2S at subcellular level. In this paper, a novel colorimetric and NIR fluorescent probe SHCy-H2S for the specific detection of H2S in lysosome over other biological thiols was designed and synthesized. The xanthene-benzothiozolium fluorophore was chosen to provide fluorescence emission maxima over 735 nm, and 2,4-dinitrophenyl group was chosen as fluorescence quenching group and specific H2S response site. Impressively, SHCy-H2S exhibited high selectivity, fast response and detection limit as low as 0.116 µM for H2S, marked obvious color changes in naked-eye and fluorescence. Specially, SHCy-H2S was capable of specifically imaging endogenous lysosomal hydrogen sulfide, providing a potential tool for exploring the function of H2S at subcellular level.

Paraquat exposure impairs porcine oocyte meiotic maturation.

Paraquat (PQ) is a heterocyclic pesticide that not only damages the testicular development and reduces the quality of semen, but also disturbs the secretion of hormones in the reproductive system. However, the effects of PQ on oocyte maturation and its toxic mechanism have not been yet fully clarified. Here we showed that PQ exposure could have toxic effects on porcine oocyte maturation. PQ exposure with 100 µM inhibited cumulus cell expansion and significantly reduced the rate of first polar body extrusion during oocyte maturation. PQ-exposed oocytes could not develop to the 2-cell and blastocyst stage. PQ exposure with 100 µM significantly increased abnormal spindle rate (65.2% ± 1.0%) and misaligned chromosome rate (63.2% ± 3.4%) compared to the control group (38.3% ± 1.0% and 38.4% ± 1.0%, respectively; P < 0.05). F-actin also exhibited reduced distribution in PQ-exposed oocytes (10.3% ± 1.0%) compared to the control group (14.4% ± 1.0%, P < 0.05). In addition, PQ exposure reduced the active mitochondria levels, but apparently increased the reactive oxygen species (ROS), rH2AX, and LC3 (autophagy marker) levels. qPCR analyses showed that PQ exposure caused the aberrant expression of genes associated with cumulus cell expansion, but did not affect the expression of apoptosis-related genes. Taken together, these results indicate that PQ exposure impaired oocyte nuclear and cytoplasmic maturation probably through oxidative stress.

Prognostic significance of sarcopenia and systemic inflammation for patients with renal cell carcinoma following nephrectomy.

Background: To clarify the prognostic effect of preoperative sarcopenia and systemic inflammation, and to develop a nomogram for predicting overall survival (OS) of patients with renal cell carcinoma (RCC) following partial or radical nephrectomy. Methods: Patients with RCC following nephrectomy from the First Affiliated Hospital of Soochow University during January 2018 to September 2020 were included in this study. The relationship between sarcopenia and inflammatory markers was identified by logistic regression analysis. Then univariable Cox regression analysis, LASSO regression analysis and multivariable Cox regression analysis were analyzed sequentially to select the independent prognostic factors. Kaplan-Meier survival curves were applied to ascertain the prognostic value. Finally, the identified independent predictors were incorporated in a nomogram, which was internally validated and compared with other methods. Results: A total of 276 patients were enrolled, and 96 (34.8%) were diagnosed with sarcopenia, which was significantly associated with neutrophil-to-lymphocyte ratio (NLR). Sarcopenia and elevated inflammation markers, i.e., NLR, platelet-to-lymphocyte ratio (PLR) and the modified Glasgow Prognostic Score (mGPS), were independent factors for determining the OS. The model had good discrimination with Concordance index of 0.907 (95% CI: 0.882-0.931), and the calibration plots performed well. Both net reclassification index (NRI) and integrated discriminant improvement (IDI) exhibited better performance of the nomogram compared with clinical stage-based, sarcopenia-based and integrated "NLR+PLR+mGPS" methods. Moreover, decision curve analysis showed a net benefit of the nomogram at a threshold probability greater than 20%. Conclusions: Preoperative sarcopenia was significantly associated with NLR. A novel nomogram with well validation was developed for risk stratification, prognosis tracking and personalized therapeutics of RCC patients.

Chromatin remodeler INO80 mediates trophectoderm permeability barrier to modulate morula-to-blastocyst transition.

Inositol requiring mutant 80 (INO80) is a chromatin remodeler that regulates pluripotency maintenance of embryonic stem cells and reprogramming of somatic cells into pluripotent stem cells. However, the roles and mechanisms of INO80 in porcine pre-implantation embryonic development remain largely unknown. Here, we show that INO80 modulates trophectoderm epithelium permeability to promote porcine blastocyst development. The INO80 protein is highly expressed in the nuclei during morula-to-blastocyst transition. Functional studies revealed that RNA interference (RNAi)-mediated knockdown of INO80 severely blocks blastocyst formation and disrupts lineage allocation between the inner cell mass and trophectoderm. Mechanistically, single-embryo RNA sequencing revealed that INO80 regulates multiple genes, which are important for lineage specification, tight junction assembly, and fluid accumulation. Consistent with the altered expression of key genes required for tight junction assembly, a permeability assay showed that paracellular sealing is defective in the trophectoderm epithelium of INO80 knockdown blastocysts. Importantly, aggregation of 8-cell embryos from the control and INO80 knockdown groups restores blastocyst development and lineage allocation via direct complementation of the defective trophectoderm epithelium. Taken together, these results demonstrate that INO80 promotes blastocyst development by regulating the expression of key genes required for lineage specification, tight junction assembly, and fluid accumulation.

Laparoscopic radical resection for large-volume renal carcinoma: via retroperitoneal or transperitoneal approach?

PURPOSE: The aim of this study was to investigate the clinical efficacy and safety of laparoscopic radical resection through retroperitoneal and transperitoneal approaches in treating large-volume renal carcinoma. METHODS: A total of 116 patients with large-volume (>7 cm) renal carcinoma underwent laparoscopic radical resection for renal carcinoma. Among them, 58 were treated through retroperitoneal approach (Retroperitoneal group), and 58 were treated through transperitoneal approach (Abdominal group). The levels of interleukin-6 (IL-6), IL-12 and IL-1ß in the patients were compared before and after operation. Furthermore, the levels of tumor markers were explored, and the tumor recurrence and survival of the patients were followed up and recorded. RESULTS: Compared with those in Abdominal group, the patients in Retroperitoneal group had remarkably shorter operation time, time of renal artery occlusion, time of intestinal exhaust and length of hospital stay after operation as well as notably smaller intraoperative blood loss. The levels of IL-6, IL-12 and IL-1ß were elevated after operation in both groups in comparison with those before operation. Besides, the concentrations of serum CA50, CA125 and CEA declined obviously after treatment in the two groups in contrast with those before treatment, while no statistically significant differences in the concentrations of serum CA50, CA125 and CEA were observed between the two groups after treatment. The follow-up results indicated that the average survival and progression-free survival were 18.3 months and 16.0 months, respectively, in Retroperitoneal group, and 19.1 months and 16.8 months, respectively, in Abdominal group. CONCLUSIONS: The retroperitoneal laparoscopic radical resection for large-volume renal carcinoma possesses exact therapeutic effects, and it has shorter operation time, less blood loss, fewer impacts on inflammatory responses in patients and higher safety than transperitoneal laparoscopic radical resection.

Histone Arginine Methyltransferase CARM1-Mediated H3R26me2 Is Essential for Morula-to-Blastocyst Transition in Pigs.

Coactivator-associated arginine methyltransferase 1 (CARM1) is involved in both establishment of first pluripotent lineage and pluripotency maintenance of embryonic stem cells (ESCs) in mice. However, the histone substrates and role of CARM1 in early embryonic development remain largely unknown. Here, we show that CARM1 specifically catalyzes H3R26me2 to promote porcine blastocyst formation. The putative histone substrates of CARM1, including H3R2me2, H3R17me2, and H3R26me2, are present in pig early embryos. The changes of CARM1 mRNA during early embryogenesis parallel that of H3R26me2. Functional studies using a combinational approach of chemical inhibition and RNA interference (RNAi) showed that catalytic activity inhibition of CARM1 protein or knockdown (KD) of CARM1 mRNA did not alter the levels of both H3R2me2 and H3R17me2, but significantly reduced H3R26me2 levels in porcine embryos. Furthermore, CARM1 inhibition or KD did not affect embryo development to the 2-cell, 4-cell, 8-cell, and morula stages, but severely compromised blastocyst development. CARM1 knocked down embryos that developed to the blastocyst stage had fewer total cells, inner cell mass (ICM), and trophectoderm (TE) cells. Mechanistically, single embryo RNA-sequencing analysis revealed that CARM1 KD altered the transcriptome characterized by downregulation of key genes associated with Hippo and PI3K-AKT signaling pathways. Taken together, these results demonstrate that CARM1 specifically catalyzes H3R26me2 in porcine embryos and participates in blastocyst development.

Impact of Diabetes Mellitus on Lower Urinary Tract Symptoms in Benign Prostatic Hyperplasia Patients: A Meta-Analysis.

Background: Benign prostatic hyperplasia (BPH) is a disease that causes lower urinary tract symptoms (LUTS), which are the most common urological problem in approximately one-third of the male population aged over 50 years. Some studies have suggested that diabetes may be a risk factor for the development of BPH. However, whether diabetes aggravates the LUTS of BPH patients is still controversial. Aim: To investigate the impact of diabetes mellitus on LUTS in BPH patients. Methods: A literature search was conducted using Web of Science, Embase, PubMed, and China National Knowledge Infrastructure literature databases. This meta-analysis was registered in PROSPERO (registration number: CRD 42020200794). Fixed- or random-effects models were used for analysis according to heterogeneity. The results of the systematic analysis are presented as weighted mean difference (WMD) with the corresponding 95% confidence intervals (CI). Results: In total, 1308 studies were retrieved from databases and 18 articles comprising 1685 cases and 4653 controls were selected for meta-analysis. The results of the meta-analysis showed that the International Prostate Symptom Score (IPSS) value and prostate volume of BPH patients with diabetes was significantly higher than that of BPH patients without diabetes. Conclusions: This systematic review is the first to evaluate the impact of diabetes mellitus on LUTS in BPH patients. The results of our meta-analysis support the hypothesis that LUTS in BPH patients is increased in patients with diabetes mellitus compared with controls, which suggests that physicians should pay more attention to BPH patients with diabetes mellitus. Systematic Review Registration: PROSPERO [https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=200794], identifier CRD 42020200794.

Resection of adult polycystic kidney with retroperitoneal laparoscopic technique assisted by arterial embolization.

BACKGROUND: Traditional surgical methods have high complication rate and large injury in the resection of adult polycystic kidney. We investigated the effect of retroperitoneal laparoscopic resection of adult polycystic kidney assisted by arterial embolization. METHODS: The data of adult polycystic kidney patients who underwent laparoscopic surgery assisted by arterial embolization from November 2015 to November 2018 in our hospital were retrospectively analyzed, and the data of patients who underwent open surgery during the same period were collected. The basic data, surgical conditions, postoperative recover situation, and complications of the two groups were compared. RESULTS: There was no significant difference in the basic situation between the laparoscopic operation group and open operation (control) group. The bleeding volume, hospitalization time, and the length of incision in the laparoscopic operation group were significantly better than those in the open operation (control) group, but the operation time was significantly longer than that in the open operation group. There was no significant difference in drainage tube extraction time, bed rest time and blood transfusion rate between the two groups. There was no significant difference in the complication rate between the two groups. CONCLUSIONS: Arterial interventional embolization-assisted retroperitoneal laparoscopy is an effective method for the resection of polycystic kidney.

Genome-Wide Survey of Invertase Encoding Genes and Functional Characterization of an Extracellular Fungal Pathogen-Responsive Invertase in Glycine max.

Invertases are essential enzymes that irreversibly catalyze the cleavage of sucrose into glucose and fructose. Cell wall invertase (CWI) and vacuolar invertase (VI) are glycosylated proteins and exert fundamental roles in plant growth as well as in response to environmental cues. As yet, comprehensive insight into invertase encoding genes are lacking in Glycine max. In the present study, the systematic survey of gene structures, coding regions, regulatory elements, conserved motifs, and phylogenies resulted in the identification of thirty⁻two putative invertase genes in soybean genome. Concomitantly, impacts on gene expression, enzyme activities, proteins, and soluble sugar accumulation were explored in specific tissues upon stress perturbation. In combination with the observation of subcellular compartmentation of the fluorescent fusion protein that indeed exported to apoplast, heterologous expression, and purification in using Pichia pastoris system revealed that GmCWI4 was a typical extracellular invertase. We postulated that GmCWI4 may play regulatory roles and be involved in pathogenic fungi defense. The experimental evaluation of physiological significance via phenotypic analysis of mutants under stress exposure has been initiated. Moreover, our paper provides theoretical basis for elucidating molecular mechanisms of invertase in association with inhibitors underlying the stress regime, and will contribute to the improvement of plant performance to a diverse range of stressors.