GDPF: a data resource for the distribution of prokaryotic protein families across the global biosphere.

Microorganisms encode most of the functions of life on Earth. However, conventional research has primarily focused on specific environments such as humans, soil and oceans, leaving the distribution of functional families throughout the global biosphere poorly comprehended. Here, we present the database of the global distribution of prokaryotic protein families (GDPF, http://bioinfo.qd.sdu.edu.cn/GDPF/), a data resource on the distribution of functional families across the global biosphere. GDPF provides global distribution information for 36 334 protein families, 19 734 superfamilies and 12 089 KEGG (Kyoto Encyclopedia of Genes and Genomes) orthologs from multiple source databases, covering typical environments such as soil, oceans, animals, plants and sediments. Users can browse, search and download the distribution data of each entry in 10 000 global microbial communities, as well as conduct comparative analysis of distribution disparities among multiple entries across various environments. The GDPF data resource contributes to uncovering the geographical distribution patterns, key influencing factors and macroecological principles of microbial functions at a global level, thereby promoting research in Earth ecology and human health.

PAT: a comprehensive database of prokaryotic antimicrobial toxins.

Antimicrobial toxins help prokaryotes win competitive advantages in intraspecific or interspecific conflicts and are also a critical factor affecting the pathogenicity of many pathogens that threaten human health. Although many studies have revealed that antagonism based on antimicrobial toxins plays a central role in prokaryotic life, a database on antimicrobial toxins remains lacking. Here, we present the prokaryotic antimicrobial toxin database (PAT, http://bioinfo.qd.sdu.edu.cn/PAT/), a comprehensive data resource collection on experimentally validated antimicrobial toxins. PAT has organized information, derived from the reported literature, on antimicrobial toxins, as well as the corresponding immunity proteins, delivery mechanisms, toxin activities, structural characteristics, sequences, etc. Moreover, we also predict potential antimicrobial toxins in prokaryotic reference genomes and show the taxonomic information and environmental distribution of typical antimicrobial toxins. These details have been fully incorporated into the PAT database, where users can browse, search, download, analyse and view informative statistics and detailed information. PAT resources have already been used in our prediction and identification of prokaryotic antimicrobial toxins and may contribute to promoting the efficient investigation of antimicrobial toxin functions, the discovery of novel antimicrobial toxins, and an improved understanding of the biological roles and significance of these toxins.

Assembly processes and source tracking of planktonic and benthic bacterial communities in the Yellow River estuary.

Estuaries connect rivers with the ocean and are considered transition regions due to the continuous inputs from rivers. Microbiota from different sources converge and undergo succession in these transition regions, but their assembly mechanisms along environmental gradients remain unclear. Here, we found that salinity had a stronger effect on planktonic than on benthic microbial communities, and the dominant planktonic bacteria changed more distinctly than the dominant benthic bacteria with changes in salinity. The planktonic bacteria in the brackish water came mainly from seawater, which was confirmed in the laboratory, whereas the benthic bacteria were weakly affected by salinity, which appeared to be a mixture of the bacteria from riverine and oceanic sediments. Benthic bacterial community assembly in the sediments was mainly controlled by homogeneous selection and almost unaffected by changes in salinity, the dominant assemblage processes for planktonic bacteria changed dramatically along the salinity gradient, from homogeneous selection in freshwater to drift in seawater. Our results highlight that salinity is the key driver of estuarine microbial succession and that salinity is more important in shaping planktonic than benthic bacterial communities in the Yellow River estuary.

High-efficiency water-window x-ray generation from nanowire array targets irradiated with femtosecond laser pulses.

We demonstrate the high-efficiency generation of water-window soft x-ray emissions from polyethylene nanowire array targets irradiated by femtosecond laser pulses at the intensity of 4×1019 W/cm2. The experimental results indicate more than one order of magnitude enhancement of the water-window x-ray emissions from the nanowire array targets compared to the planar targets. The highest energy conversion efficiency from laser to water-window x-rays is measured as 0.5%/sr, which comes from the targets with the longest nanowires. Supported by particle-in-cell simulations and atomic kinetic codes, the physics that leads to the high conversion efficiency is discussed.

High-efficiency generation of narrowband soft x rays from carbon nanotube foams irradiated by relativistic femtosecond lasers.

A number of applications require x rays of both high flux and narrow bandwidth. In this work, we experimentally demonstrate the high-efficiency generation of narrowband soft x rays from carbon nanotube foams irradiated by a femtosecond laser pulse at an intensity of 1019W/cm2. The building blocks of the foam, single-walled carbon nanotube bundles with diameters smaller than the laser skin length can be volumetrically heated and fully ionized on a femtosecond time scale. The three-dimensional network structure of the foam permits deep penetration and drastic absorption of the laser pulse, and results in bright line emissions without prominent Stark broadening. A single-shot yield of 3×1014photons in the carbon Lyα line at 3.37 nm was measured with a bandwidth of 0.013 nm.

Optogenetic Strategies for Vision Restoration.

The loss of photoreceptor cells caused by retinal degenerative diseases leads to blindness. The optogenetic approach for restoring vision involves converting the surviving inner retinal neurons into photosensitive cells, thus imparting light sensitivity to the retina following the loss of photoreceptor cells. Our first demonstration of the feasibility of such an approach involved expressing ChR2 in the retinal ganglion cells of blind mice; since then, optogenetic vision restoration has been demonstrated by using a variety of optogenetic tools, especially microbial channelrhodopsins (ChRs). A ChR-based optogenetic therapy for treating blindness has advanced to clinical trials. In this chapter, we review our early proof-of-concept study of optogenetic vision restoration. We also discuss our studies for developing better ChR tools and for restoring intrinsic visual processing features in retinas with degenerated photoreceptors.

Improved CoChR Variants Restore Visual Acuity and Contrast Sensitivity in a Mouse Model of Blindness under Ambient Light Conditions.

Severe photoreceptor cell death in retinal degenerative diseases leads to partial or complete blindness. Optogenetics is a promising strategy to treat blindness. The feasibility of this strategy has been demonstrated through the ectopic expression of microbial channelrhodopsins (ChRs) and other genetically encoded light sensors in surviving retinal neurons in animal models. A major drawback for ChR-based visual restoration is low light sensitivity. Here, we report the development of highly operational light-sensitive ChRs by optimizing the kinetics of a recently reported ChR variant, Chloromonas oogama (CoChR). In particular, we identified two CoChR mutants, CoChR-L112C and CoChR-H94E/L112C/K264T, with markedly enhanced light sensitivity. The improved light sensitivity of the CoChR mutants was confirmed by ex vivo electrophysiological recordings in the retina. Furthermore, the CoChR mutants restored the vision of a blind mouse model under ambient light conditions with remarkably good contrast sensitivity and visual acuity, as evidenced by the results of behavioral assays. The ability to restore functional vision under normal light conditions with the improved CoChR variants removed a major obstacle for ChR-based optogenetic vision restoration.

Sex-related differences in the progressive retinal degeneration of the rd10 mouse.

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6ß proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

[Research progress of 5-hydroxymethylfurfural, a safety-related substance in traditional Chinese medicine injections].

Screening out the safety-related substances and establishing the corresponding standard has been a key research issue to improve the safety of traditional Chinese medicine injections(TCMIs). 5-HMF which widely exists in sugar-containing TCMIs has long been considered as an important safety-related substance. In this review, we summarizes the research progress on the toxicology of 5-HMF as well as the content and standards of 5-HMF in TCMIs.Therein, both literature summary and analysis results indicate that there are lack of toxicology researches of 5-HMF and its metabolites in TCMIs, although the potential toxicity of 5-HMF and its metabolites has been reported. Moreover, the content of 5-HMF largely varies from TCMIs to TCMIs, and even in the same TCMIs from different factories. To ensure the clinical efficacy of TCMIs, it urgent to carry out the study of the toxicology of 5-HMF in TCMIs comprehensively and systematically, so as to set up a relatively uniform standard as well as to develop process quality control method.

Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus.

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.

Isolation and molecular typing of Cronobacter spp. in commercial powdered infant formula and follow-up formula.

Cronobacter spp. (Enterobacter sakazakii) are important foodborne pathogens. Infections with this pathogen can lead to neonatal meningitis, necrotizing enterocolitis, and bacteremia. This study examined Cronobacter spp. contamination in commercial powdered infant formulas (PIFs) and follow-up formulas (FUFs) in China. Forty-nine of 399 samples were contaminated with Cronobacter spp. and 10.2% of the isolates were resistant to cefotaxime; in contrast, all of the tested isolates were susceptible to amikacin, amoxicillin/clavulanic acid, cefepime, ciprofloxacin, imipenem, and meropenem. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) analyses produced a total of 16 PFGE banding patterns and 11 sequence types (STs), including 7 novel STs. In summary, the rates at which Cronobacter spp. were isolated from commercial PIF and FUF samples in China were relatively high, and the isolated strains exhibited high susceptibility in vitro to most antibiotics. The PFGE method exhibited higher typing capability than the MLST method, and molecular typing results revealed that the contamination of PIF and FUF with Cronobacter spp. in China may be mainly due to the addition of contaminated materials. Thus, the development of more effective control strategies during the manufacturing process is needed.

Integrative transcriptomic and proteomic profile revealed inhibition of oxidative phosphorylation and peroxisomes during renal interstitial fibrosis.

Effective therapies of chronic kidney disease (CKD) are lacking due to the unclear molecular pathogenesis. Previous single omics-studies have described potential molecular regulation mechanism of CKD only at the level of transcription or translation. Therefore, this study generated an integrated transcriptomic and proteomic profile to provide deep insights into the continuous transcription-translation process during CKD. The comprehensive datasets identified 14,948 transcripts and 6423 proteins, 233 up-regulated and 364 down-regulated common differentially expressed genes of transcriptome and proteome were selected to further combined bioinformatics analysis. The obtained results revealed reactive oxygen species (ROS) metabolism and antioxidant system due to imbalance of mitochondria and peroxisomes were significantly repressed in CKD. Overall, this study presents a valuable multi-omics analysis that sheds light on the molecular mechanisms underlying CKD. SIGNIFICANCE: Chronic kidney disease (CKD) is a progressive and irreversible condition that results in abnormal kidney function and structure, and is ranked 18th among the leading causes of death globally, leading to a significant societal burden. Hence, there is an urgent need for research to detect new, sensitive, and specific biomarkers. Omics-based studies offer great potential to identify underlying disease mechanisms, aid in clinical diagnosis, and develop novel treatment strategies for CKD. Previous studies have mainly focused on the regulation of gene expression or protein synthesis in CKD, thereby compelling us to conduct a meticulous analysis of transcriptomic and proteomic data from the UUO mouse model. Here, we have performed a unified analysis of CKD model by integrating transcriptomes and protein suites for the first time. Our study contributes to a deeper understanding of the pathogenesis of CKD and provides a basis for subsequent disease management and drug development.

The value of G-CSF in women experienced at least one implantation failure: a systematic review and meta-analysis.

Objective: Despite the developments of in vitro fertilization (IVF) protocols, implantation failure remains a challenging problem, owing to the unbalance between the embryo, endometrium, and immune system interactions. Effective treatments are urgently required to improve successful implantation. Recently, many researchers have focused on granulocyte colony-stimulating factor (G-CSF) to regulate immune response and embryo-endometrium cross-talk. However, previous studies have reported inconsistent findings on the efficacy of G-CSF therapy on implantation failure. The objective of this review was to further explore the effects of G-CSF according to administration dosage and timing among women who experienced at least one implantation failure. Methods: We systematically searched MEDLINE, Embase, the Cochrane Central Register of Controlled Trials, Scopus, and Web of Science for randomized controlled trials of G-CSF on implantation failure up to July 21, 2023. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated and the heterogeneity of the studies with the I2 index was analyzed. Results: We identified a total of 2031 studies and finally included 10 studies in the systematic review and meta-analysis. G-CSF administration improved the clinical pregnancy rate (CPR), implantation rate (IR), biochemical pregnancy rate (BPR), and live birth rate (LBR) in women with at least one implantation failure. Subgroup analyses showed that G-CSF treatment could exert good advantages in improving CPR [OR=2.49, 95%CI (1.56, 3.98), I2 = 0%], IR [OR=2.82, 95%CI (1.29, 6.15)], BPR [OR=3.30, 95%CI (1.42, 7.67)] and LBR [OR=3.16, 95%CI (1.61, 6.22), I2 = 0%] compared with the blank control group. However, compared with placebo controls, G-CSF showed beneficial effects on CPR [OR=1.71, 95%CI (1.04, 2.84), I2 = 38%] and IR [OR=2.01, 95%CI (1.29, 3.15), I2 = 24%], but not on LBR. In addition, >150µg of G-CSF treatment increased CPR [OR=2.22, 95%CI (1.47, 3.35), I2 = 0%], IR [OR=2.67, 95%CI (1.47, 4.82), I2 = 0%] and BPR [OR=2.02, 95%CI (1.17, 3.47), I2 = 22%], while ≤150µg of G-CSF treatment improved miscarriage rate (MR) [OR=0.14, 95%CI (0.05, 0.38), I2 = 0%] and LBR [OR=2.65, 95%CI (1.56, 4.51), I2 = 0%]. Moreover, G-CSF administration on the day of embryo transfer (ET) could increase CPR [OR=2.81, 95%CI (1.37, 5.75), I2 = 0%], but not on the day of ovum pick-up (OPU) or human chorionic gonadotropin (HCG) injection. Conclusion: G-CSF has a beneficial effect on pregnancy outcomes to some extent among women who experienced at least one implantation failure, and the administration dosage and timing influence the effect size.Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42023447046.

DnaK duplication and specialization in bacteria correlates with increased proteome complexity.

The chaperone 70 kDa heat shock protein (Hsp70) is important for cells from bacteria to humans to maintain proteostasis, and all eukaryotes and several prokaryotes encode Hsp70 paralogs. Although the mechanisms of Hsp70 function have been clearly illuminated, the function and evolution of Hsp70 paralogs is not well studied. DnaK is a highly conserved bacterial Hsp70 family. Here, we show that dnaK is present in 98.9% of bacterial genomes, and 6.4% of them possess two or more DnaK paralogs. We found that the duplication of dnaK is positively correlated with an increase in proteomic complexity (proteome size, number of domains). We identified the interactomes of the two DnaK paralogs of Myxococcus xanthus DK1622 (MxDnaKs), which revealed that they are mostly nonoverlapping, although both prefer α and ß domain proteins. Consistent with the entire M. xanthus proteome, MxDnaK substrates have both significantly more multi-domain proteins and a higher isoelectric point than that of Escherichia coli, which encodes a single DnaK homolog. MxDnaK1 is transcriptionally upregulated in response to heat shock and prefers to bind cytosolic proteins, while MxDnaK2 is downregulated by heat shock and is more associated with membrane proteins. Using domain swapping, we show that the nucleotide-binding domain and the substrate-binding ß domain are responsible for the significant differences in DnaK interactomes, and the nucleotide binding domain also determines the dimerization of MxDnaK2, but not MxDnaK1. Our work suggests that bacterial DnaK has been duplicated in order to deal with a more complex proteome, and that this allows evolution of distinct domains to deal with different subsets of target proteins.IMPORTANCEAll eukaryotic and ~40% of prokaryotic species encode multiple 70 kDa heat shock protein (Hsp70) homologs with similar but diversified functions. Here, we show that duplication of canonical Hsp70 (DnaK in prokaryotes) correlates with increasing proteomic complexity and evolution of particular regions of the protein. Using the Myxococcus xanthus DnaK duplicates as a case, we found that their substrate spectrums are mostly nonoverlapping, and are both consistent to that of Escherichia coli DnaK in structural and molecular characteristics, but show differential enrichment of membrane proteins. Domain/region swapping demonstrated that the nucleotide-binding domain and the ß substrate-binding domain (SBDß), but not the SBDα or disordered C-terminal tail region, are responsible for this functional divergence. This work provides the first direct evidence for regional evolution of DnaK paralogs.

Determination of the chromosomal position effects for plug-and-play application in the Myxococcus xanthus chassis cells.

The chromosomal position effect can significantly affect the transgene expression, which may provide an efficient strategy for the inauguration of alien genes in new hosts, but has been less explored rationally. The bacterium Myxococcus xanthus harbors a large circular high-GC genome, and the position effect in this chassis may result in a thousand-fold expression variation of alien natural products. In this study, we conducted transposon insertion at TA sites on the M. xanthus genome, and used enrichment and dilution indexes to respectively appraise high and low expression potentials of alien genes at insertion sites. The enrichment sites are characteristically distributed along the genome, and the dilution sites are overlapped well with the horizontal transfer genes. We experimentally demonstrated the enrichment sites as high expression integration sites (HEISs), and the dilution sites unsuitable for gene integration expression. This work highlights that HEISs are the plug-and-play sites for efficient expression of integrated genes.

Functional investigation of the two ClpPs and three ClpXs in Myxococcus xanthus DK1622.

ClpXP is a protease complex that plays important roles in protein quality control and cell cycle regulation, but the functions of multiple ClpXs and multiple ClpPs in M. xanthus remain unknown. The genome of Myxococcus xanthus DK1622 contains two clpPs and three clpXs. The clpP1 and clpX1 genes are cotranscribed and are both essential, while the other copies are isolated in the genome and are deletable. The deletion of clpX2 caused the mutant to be deficient in fruiting body development, while the clpX3 gene is involved in resistance to thermal stress. Both ClpPs possess catalytic active sites, but only ClpP1 shows in vitro peptidase activity on the typical substrate Suc-LY-AMC. All of these clpP and clpX genes exhibit strong transcriptional upregulation in the stationary phase, and the transcription of the three clpX genes appears to be coordinated. Our results demonstrated that multiple ClpPs and multiple ClpXs are functionally divergent and may assist in the environmental adaptation and functional diversification of M. xanthus.IMPORTANCEClpXP is an important protease complex of bacteria and is involved in various physiological processes. Myxococcus xanthus DK1622 possesses two ClpPs and three ClpXs with unclear functions. We investigated the functions of these genes and demonstrated the essential roles of clpP1 and clpX1. Only ClpP1 has in vitro peptidase activity on Suc-LY-AMC, and the isolated clpX copies participate in distinct cellular processes. All of these genes exhibited significant transcriptional upregulation in the stationary phase. Divergent functions appear in multiple ClpPs and multiple ClpXs in M. xanthus DK1622.

Deciphering the Biosynthesis and Physiological Function of 5-Methylated Pyrazinones Produced by Myxobacteria.

Myxobacteria are a prolific source of secondary metabolites with sheer chemical complexity, intriguing biosynthetic enzymology, and diverse biological activities. In this study, we report the discovery, biosynthesis, biomimetic total synthesis, physiological function, structure-activity relationship, and self-resistance mechanism of the 5-methylated pyrazinone coralinone from a myxobacterium Corallococcus exiguus SDU70. A single NRPS/PKS gene corA was genetically and biochemically demonstrated to orchestrate coralinone, wherein the integral PKS part is responsible for installing the 5-methyl group. Intriguingly, coralinone exacerbated cellular aggregation of myxobacteria grown in liquid cultures by enhancing the secretion of extracellular matrix, and the 5-methylation is indispensable for the alleged activity. We provided an evolutionary landscape of the corA-associated biosynthetic gene clusters (BGCs) distributed in the myxobacterial realm, revealing the divergent evolution for the diversity-oriented biosynthesis of 5-alkyated pyrazinones. This phylogenetic contextualization provoked us to identify corB located in the proximity of corA as a self-resistance gene. CorB was experimentally verified to be a protease that hydrolyzes extracellular proteins to antagonize the agglutination-inducing effect of coralinone. Overall, we anticipate these findings will provide new insights into the chemical ecology of myxobacteria and lay foundations for the maximal excavation of these largely underexplored resources.

Generation of attosecond gigawatt soft x-ray pulses through coherent Thomson backscattering.

Collision between relativistic electron sheets and counterpropagating laser pulses is recognized as a promising way to produce intense attosecond x rays through coherent Thomson backscattering (TBS). In a double-layer scheme, the electrons in an ultrathin solid foil are first pushed out by an intense laser driver and then interact with the laser reflected off a second foil to form a high-density relativistic electron sheet with vanishing transverse momentum. However, the repulsion between these concentrated electrons can increase the thickness of the layer, reducing both its density and subsequently the coherent TBS. Here, we present a systematic study on the evolution of the flying electron layer and find that its resulting thickness is determined by the interplay between the intrinsic space-charge expansion and the velocity compression induced by the drive laser. How the laser driver, the target areal density, the reflector, and the collision laser intensity affect the properties of the produced x rays is explored. Multidimensional particle-in-cell simulations indicate that employing this scheme in the nonlinear regime has the potential to stably produce soft x rays with several gigawatt peak power in hundreds of terawatt ultrafast laser facilities. The pulse duration can be tuned to tens of attoseconds. This compact and intense attosecond x-ray source may have broad applications in attosecond science.

Elevated RHAMM as a biomarker for predicting diabetic kidney disease in patients with type 2 diabetes.

Background: Diabetic kidney disease (DKD) poses a significant challenge globally as a complication of diabetes. Hyaluronan (HA), a critical non-sulfated glycosaminoglycan in the extracellular matrix, plays a pivotal role in the progression of DKD. This study assesses the predictive significance of HA's corresponding receptor, RHAMM (receptor for HA-mediated motility), in DKD pathogenesis in type 2 diabetes (T2DM) patients. Methods: Enzyme-linked immunosorbent assays were utilized to measure plasma and urine levels of HA, CD44 and RHAMM in 99 diabetic patients. Immunohistochemistry staining was employed to examine HA deposition, CD44 and RHAMM expressions from 18 biopsy-proven DKD patients. Spearman correlation analysis, linear regression and receiver operating characteristic (ROC) analysis were conducted to establish associations between plasma HA, CD44 and RHAMM levels, and clinical parameters in DKD patients with T2DM. Results: Elevated plasma and urine HA, CD44 and RHAMM levels were notably observed in the severe renal dysfunction group. Plasma RHAMM exhibited positive correlations with HA (r = 0.616, P < .001) and CD44 (r = 0.220, P < .001), and a negative correlation with estimated glomerular filtration rate (eGFR) (r = -0.618, P < .001). After adjusting for other potential predictors, plasma RHAMM emerged as an independent predictor of declining eGFR (ß = -0.160, P < .05). Increased HA, CD44 and RHAMM levels in kidney biopsies of DKD patients were closely associated with heightened kidney injury. The ROC curve analysis highlighted an area under the curve (AUC) of 0.876 for plasma RHAMM, indicating superior diagnostic efficacy compared to CD44 in predicting DKD pathogenesis. The combined AUC of 0.968 for plasma RHAMM, HA and CD44 also suggested even greater diagnostic potential for DKD pathogenesis. Conclusion: These findings provide initial evidence that elevated RHAMM levels predict DKD pathogenesis in T2DM patients. The formation of a triple complex involving HA, CD44 and RHAMM on the cell surface shows promise as a targetable biomarker for early intervention to mitigate severe renal dysfunctions.

Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines.

PURPOSE: Retinal bipolar cells, comprising multiple types, play an essential role in segregating visual information into multiple parallel pathways in the retina. The ability to manipulate gene expression in specific bipolar cell type(s) in the retina is important for understanding the molecular basis of their normal physiological functions and diseases/disorders. The Cre/LoxP recombination system has become an important tool for allowing gene manipulation in vivo, especially with the increasing availability of cell- and tissue-specific Cre transgenic mouse lines. Detailed in vivo examination of the Cre/LoxP recombination efficiency and the transgene expression patterns for cell- and tissue-specific Cre transgenic mouse lines is essential for evaluating their utility. In this study, we investigated the Cre-mediated recombination efficiency and transgene expression patterns of retinal bipolar cell-expressing Cre transgenic lines by crossing with a Cre reporter mouse line and through Cre-dependent recombinant adeno-associated virus (rAAV) vector-mediated transgene delivery. METHODS: Three retinal bipolar cell-expressing Cre-transgenic mouse lines, 5-HTR2a-cre, Pcp2-cre, and Chx10-cre, were crossed with a strong Cre reporter mouse line that expresses a red fluorescent protein variant, tdTomato. rAAV2 vectors carrying a double-floxed inverted open-reading frame sequence encoding channelrhodopsin-2-mCherry (ChR2-mCherry) driven by a ubiquitous neuronal EF1α or a ubiquitous CMV promoter with a rAAV2 capsid mutation (Y444F) were injected into the intravitreal space of the eyes. Immunohistochemistry using retinal bipolar cell type-specific markers was performed to examine Cre-mediated recombination efficiency and the transgene expression patterns in bipolar cells in retinal whole mounts and vertical sections. RESULTS: For the 5-HTR2a-cre and Pcp2-cre mouse lines, the expression pattern of the Cre-mediated recombination by crossing the reporter line largely resembled the expression pattern of Cre. The bipolar cells showing Cre-mediated recombination in the 5-HTR2a-cre line and the Pcp2-cre line were predominantly type 4 cone bipolar cells and rod bipolar cells, respectively. For the Chx10-cre mouse line, the expression pattern of the Cre-mediated recombination by crossing the reporter line was different from that of Cre. The Cre-mediated transgene expression in retinal bipolar cells in the Chx10-cre line was not observed by crossing with the reporter mouse line but through Cre-dependent rAAV vector delivery. A rAAV2 vector with the combination of a CMV promoter and the Y444F capsid mutation achieved Cre-dependent transgene expression in retinal bipolar cells. CONCLUSIONS: The retinal bipolar cell-expressing Cre-transgenic lines and the Cre-dependent rAAV vector reported in this study could be valuable tools for gene targeting and manipulation in retinal bipolar cells in mice.