Molecular identification and phylogenetic analysis of gastrointestinal nematodes in different populations of Kazakh sheep.

Gastrointestinal nematode (GIN) infection in sheep has been recognized globally as a major problem challenging animal health and production. The objective of this study is to use a molecular diagnosis of the prevalence for gastrointestinal nematode (GIN) dominant species of Kazakh sheep and its hybrid (Kazakh × Texel). The internal transcribed spacer 2 (ITS-2) sequences of ribosomal DNA (rDNA) were used as the target sequence. In the study, three dominant species of nematodes, namely Haemonchus contortus, Trichostrongylus spp., and Teladorsagia (Ostertagia) circumcincta from the Kazakh sheep and the F1 and F2 generations of Texel × Kazakh sheep hybrids were subjected to molecular identification and phylogenetic analysis. The fecal and single larva genomic DNA were extracted and amplified by PCR using specific primers to determine the infection rate of the three nematode species. In addition, the PCR products were sequenced and analyzed using bioinformatics methods to construct a phylogenetic tree. The results showed that all the three species had their ITS-2 specific amplified. According to the sequence homology analysis of PCR products, the results showed a high homology (above 98.5% homology) with H. contortus, Trichostrongylus spp., T. circumcincta ITS-2 sequences in GenBank. Phylogenetic analysis showed that the ITS-2 sequences of the three species were on the same branch as the ITS-2 sequences of the same species in NCBI. And on different branches from those of the ITS-2 sequences of different families, genera and species. Sequences carried out on three species from different samples showed a close relationship and little genetic difference in phylogenetic tree. The infection rates based on fecal DNA were 35.59, 25.55, and 11.24% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. While the infection rates based on larva DNA, were 24.07, 18.89, and 13.26% for H. contortus, Trichostrongylus spp., and T. circumcincta, respectively. The seasonal prevalence of the three dominant species in spring was significantly higher than that in autumn and winter. And there was no significant difference between Kazakh, F1 and F2 sheep considering the infection rate of the studied three species of nematodes. This study provides valuable molecular approaches for epidemiological surveillance and for assisting in the control of Nematodirus infection in sheep.

Effects of exogenous GnRH administration on lambing performance of oestrus-synchronized Kazak ewes during the breeding season.

The objective of this study was to investigate the effects of GnRH at insemination on lambing performance of Kazak ewes. Oestrus was synchronized in 774 adult Kazak ewes using an intravaginal sponge impregnated with flurogestone acetate. The sponge was left in the vagina for 12 days followed with an injection of 330 IU of eCG at sponge removal. Each ewe was inseminated twice at 48 h and 60 h after sponge removal. The treatment group (n = 387) was intramuscularly injected at the first insemination with a dose of 25 µg GnRH and the control group (n = 387) with saline solution. The results showed that GnRH administration significantly decreased the fertility rate. In addition, significant differences were observed between the two groups in terms of the twin birth rate, the abortion rate and the litter size. In conclusion, GnRH administration at insemination was not recommended in Kazak ewes undergoing the fixed time artificial insemination during the breeding season. The breed may be a critical determinant of the potential for the exploitation of GnRH application in sheep breeding programmes.

Potential role for human menopausal gonadotropin in ovine superovulatory regimens during the breeding season.

Human menopausal gonadotrophin (hMG) has been reported to produce a comparable superovulatory response to that of follicle stimulating hormone (FSH). Furthermore, hMG has a long half-life as compared with FSH. The present study was designed to compare hMG administered once daily and FSH administered twice daily over a 4 - day period on superovulatory response of Suffolk ewes. During the mid-luteal phase, twenty-four Suffolk donor ewes received intravaginal sponges at day 0 for 12 days. The superovulatory regimens in the Control group (n = 12) and the Treatment group (n = 12) consisted of eight injections of FSH given at twice daily and four injections of hMG given at once daily, respectively. At day 13, the donor ewes were subjected to laparoscopic insemination. Embryos were recovered, classified, and transferred to recipient ewes at day 19. Pregnancy status was determined by ultrasound examination 40 days after transfer. Lambing rate was calculated after all the ewes had delivered. No significant differences were observed between the two groups in terms of the structures recovered, transferable embryos, degenerated embryos, unfertilized oocytes, pregnancy rate and lambing rate. The results showed that once daily injection of hMG can produce a comparable superovulatory response and embryo transfer outcomes to those obtained by twice daily injection of FSH over a 4 - day period. It is feasible that hMG is used to replace FSH and reduce the number of injection treatments in ovine superovulatory regimens.

Using single-worm RNA sequencing to study C. elegans responses to pathogen infection.

BACKGROUND: Caenorhabditis elegans is an excellent research model whose populations have been used in many studies to address various biological questions. Although worm-to-worm phenotypic variations in isogenic populations have been persistently observed, they are not well understood and are often ignored or averaged out in studies, masking the impacts of such variations on data collection and interpretation. Single-worm RNA sequencing that profiles the transcriptomes of individual animals has the power to examine differences between individuals in a worm population, but this approach has been understudied. The integrity of the starting RNA, the quality of the library and sequence data, as well as the transcriptome-profiling effectiveness of single-worm RNA-seq remain unclear. Therefore, more studies are needed to improve this technique and its application in research. RESULTS: In this study, we aimed to develop a single-worm RNA-seq method that includes five steps: worm lysis and RNA extraction, cDNA synthesis, library preparation, sequencing, and sequence data analysis. We found that the mechanical lysis of worms using a Qiagen TissueLyser maintained RNA integrity and determined that the quality of our single-worm libraries was comparable to that of standard RNA-seq libraries based on assessments of a variety of parameters. Furthermore, analysis of pathogen infection-induced gene expression using single-worm RNA-seq identified a core set of genes and biological processes relating to the immune response and metabolism affected by infection. These results demonstrate the effectiveness of our single-worm RNA-seq method in transcriptome profiling and its usefulness in addressing biological questions. CONCLUSIONS: We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and have applied this method to study C. elegans responses to pathogen infection. Key aspects of our single-worm RNA-seq libraries were comparable to those of standard RNA-seq libraries. The single-worm method captured the core set of, but not all, infection-affected genes and biological processes revealed by the standard method, indicating that there was gene regulation that is not shared by all individuals in a population. Our study suggests that combining single-worm and standard RNA-seq approaches will allow for detecting and distinguishing shared and individual-specific gene activities in isogenic populations.

Prolonging the time of semen deposition increases the pregnancy rates of ewes subjected to fixed time cervical insemination during the breeding season.

The main limiting factor of artificial cervical insemination in ewes is the long and narrow fibrous cervical canal, which impedes the transport of spermatozoa and leads to lower pregnancy rates. The hypothesis that prolonging the time of semen deposition during ovine cervical insemination can increase pregnancy rates was investigated in this study. Estrus was synchronized in 150 multiparous Ujimqin ewes using a polyurethane intravaginal sponge impregnated with 45 mg of flurogestone acetate. The sponge was left in the vagina for 12 days followed by an injection of 330 IU of eCG at sponge removal. After the exclusion of two ewes due to sponge loss, the remaining 148 ewes were divided into the Treatment group (n = 75) and the Control group (n = 73). Each ewe was inseminated once between 56 and 60 h after the removal of sponges, using a new type of insemination device containing 0.25 ml of diluted semen. Semen was collected from eight Black Suffolk rams and all the ejaculates were pooled and diluted in ultra-high temperature-treated commercial skimmed milk. The time of semen deposition was prolonged to 60 s in the Treatment group, while ewes were given traditional insemination in the Control group. Pregnancy status was determined by transabdominal ultrasound examination 45 days after insemination. Lambing performance was calculated after all the ewes had been delivered. Significant differences were observed between the Treatment group and the Control group in terms of the pregnancy rate and the fecundity rate (73.3% and 93.3% vs 56.2% and 71.2%, p < .05 and p < .01, respectively). In conclusion, prolonging the time of semen deposition significantly increased pregnancy and fecundity rates in estrus-synchronized Ujimqin ewes subjected to fixed time cervical insemination.

Oxytocin increases pregnancy rates after fixed time artificial insemination in Kazak ewes.

It is probable that reduced pregnancy rates in ewes after fixed time artificial insemination (FTAI) is attributable, in part, to the reduced number of normal spermatozoa that colonize the oviduct. Administration of oxytocin stimulates both cervical dilation and uterine/oviductal contractility. The hypothesis that oxytocin can enhance sperm transport into the uteri and the oviducts, and thereby increase pregnancy rates, was tested in the present study. Oestrus was synchronized in 199 multiparous Kazak ewes using intravaginal flurogestone-impregnated sponge. The sponge was left in the vagina for 12 days followed with an injection of 330 IU of eCG at sponge removal. Each ewe was intracervically inseminated twice at 50 hr and 62 hr after the removal of sponges using an insemination catheter containing 0.25 ml of diluted semen. Semen was collected from seven Texel rams and all the ejaculates were pooled and diluted in ultra-high temperature-treated commercial skimmed milk without (Control group, 0.05 ml of saline per mL milk, n = 144) or with oxytocin supplement (Oxytocin group, 0.5 U of oxytocin per ml milk, n = 55). Pregnancy status was determined by transabdominal ultrasound examination 45 days after insemination. Lambing performance was recorded at delivery. Significant differences were observed between the Oxytocin group and the Control group in terms of the pregnancy rate and the fecundity rate (85.5% and 92.7% versus 68.8% and 72.9%, respectively). In conclusion, low dose oxytocin supplementation of semen extender significantly increased pregnancy and fecundity rates in oestrus-synchronized Kazak ewes after FTAI.

Performing IVIM-DWI using the multifunctional nanosystem for the evaluation of the antitumor microcirculation changes.

OBJECTIVES: There is a controversy about the D* and f values of intravoxel incoherent motion diffusion-weighted imaging (IVIM-DWI) for mid- and long-term efficacy monitoring of tumor blood perfusion. To monitor the antitumor efficacy of the F/A-PLGA@DOX/SPIO nanosystem via IVIM-DWI and to explore the value of parameters pseudo-diffusion (D*) and fraction of pseudo-diffusion (f) for evaluating therapeutic effect in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Thirty-six A549 tumor-bearing mice were divided randomly into three groups (each n = 12). Group 1 (G1) was injected with saline (the control group). Group 2 (G2) and group 3(G3) were injected with DOX and F/A-PLGA@DOX/SPIO, respectively. Each group underwent IVIM-DWI scanning at baseline and 3, 14, 21, and 28 days after treatment. D* and f values were derived using GE AW 4.5 post-processing station. All mice were sacrificed for pathological examination. RESULTS: The D* value of all three groups showed an upward trend, with the highest increase in G1 and the lowest in G3. Conversely, the f value of all groups trended to decrease within 7 days, of which G3 showed the most significant decline. Immunohistochemical staining revealed that vascular endothelial growth factor (VEGF)-positive staining rate and the microvessel density (MVD) of the tumors in G3 were significantly lower than those of the other groups (P < 0.05). The D* and f values were significantly and positively correlated to CD31 (r = 0.654, P < 0.001; r = 0.712, P < 0.001) and VEGF (r = 0.694, P < 0.001; r = 0.664, P < 0.001). CONCLUSION: IVIM-DWI-derived parameters D* and f are valuable indicators for the evaluation of the antitumor microcirculation changes of multifunctional nanosystem.

Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is caused by a point mutation in the LMNA gene, resulting in production of a truncated farnesylated-prelamin A protein (progerin). We previously reported that XPA mislocalized to the progerin-induced DNA double-strand break (DSB) sites, blocking DSB repair, which led to DSB accumulation, DNA damage responses, and early replication arrest in HGPS. In this study, the XPA mislocalization to DSBs occurred at stalled or collapsed replication forks, concurrent with a significant loss of PCNA at the forks, whereas PCNA efficiently bound to progerin. This PCNA sequestration likely exposed ds-ssDNA junctions at replication forks for XPA binding. Depletion of XPA or progerin each significantly restored PCNA at replication forks. Our results suggest that although PCNA is much more competitive than XPA in binding replication forks, PCNA sequestration by progerin may shift the equilibrium to favor XPA binding. Furthermore, we demonstrated that progerin-induced apoptosis could be rescued by XPA, suggesting that XPA-replication fork binding may prevent apoptosis in HGPS cells. Our results propose a mechanism for progerin-induced genome instability and accelerated replicative senescence in HGPS.-Hilton, B. A., Liu, J., Cartwright, B. M., Liu, Y., Breitman, M., Wang, Y., Jones, R., Tang, H., Rusinol, A., Musich, P. R., Zou, Y. Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy-related progeroid syndromes.

Organismal regulation of XBP-1-mediated unfolded protein response during development and immune activation.

The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1--a G protein-coupled catecholamine receptor expressed in neurons--suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.

Contrast-enhanced CT in the differential diagnosis of bladder cancer and paraganglioma.

PURPOSE: We sought to summarize the value of contrast-enhanced computed tomography (CECT) in the differential diagnosis of bladder paraganglioma (BPG) and bladder cancer. METHODS: The medical records of 19 patients with BPG (13 males, 6 females) and 56 patients with bladder cancer (49 males, 7 females) between November 2007 and June 2023 were retrospectively reviewed. All patients underwent unenhanced and contrast-enhanced CT scanning. RESULTS: Patient age (46.4 ± 11.1 years vs. 58.6 ± 16.0 years), tumor calcification (1/19 vs. 18/56), stalk (0/19 vs. 10/56), internal vessels (15/19 vs. 19/56) and the enlarged adjacent supplying artery (14/19 vs. 10/56) were significantly different between BPG and bladder cancer (P < 0.05). The CT value in the corticomedullary phase (92.4 ± 16.6 HU vs. 64.0 ± 14.5 HU) and the contrast-enhanced value in the corticomedullary phase (54.5 ± 17.4 HU vs. 28.5 ± 12.8 HU) were significantly greater in BPG patients than in bladder cancer patients (P < 0.001), with corresponding area under the curve values of 0.930 and 0.912, respectively. The optimal cutoff values were 83.2 HU and 38.5 HU, respectively. A CT value > 83.2 HU in the corticomedullary phase and a contrast-enhanced CT value > 38.5 HU in the corticomedullary phase were used to indicate BPG with sensitivities of 78.9% and 89.5%, respectively, and specificities of 94.6% and 75.0%, respectively. CONCLUSION: The corticomedullary phase of CECT plays an important role in the preoperative differential diagnosis of BPG and bladder cancer.

PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance.

The DNA mismatch repair protein PMS2 was recently found to encode a novel endonuclease activity. To determine the biological functions of this activity in mammals, we generated endonuclease-deficient Pms2E702K knock-in mice. Pms2EK/EK mice displayed increased genomic mutation rates and a strong cancer predisposition. In addition, class switch recombination, but not somatic hypermutation, was impaired in Pms2EK/EK B cells, indicating a specific role in Ig diversity. In contrast to Pms2-/- mice, Pms2EK/EK male mice were fertile, indicating that this activity is dispensable in spermatogenesis. Therefore, the PMS2 endonuclease activity has distinct biological functions and is essential for genome maintenance and tumor suppression.

The longevity response to warm temperature is neurally controlled via the regulation of collagen genes.

Studies in diverse species have associated higher temperatures with shorter lifespan and lower temperatures with longer lifespan. These inverse effects of temperature on longevity are traditionally explained using the rate of living theory, which posits that higher temperatures increase chemical reaction rates, thus speeding up the aging process. Recent studies have identified specific molecules and cells that affect the longevity response to temperature, indicating that this response is regulated, not simply thermodynamic. Here, we demonstrate that in Caenorhabditis elegans, functional loss of NPR-8, a G protein-coupled receptor related to mammalian neuropeptide Y receptors, increases worm lifespan at 25°C but not at 20°C or 15°C, and that the lifespan extension at 25°C is regulated by the NPR-8-expressing AWB and AWC chemosensory neurons as well as AFD thermosensory neurons. Integrative transcriptomic analyses revealed that both warm temperature and old age profoundly alter gene expression and that genes involved in the metabolic and biosynthetic processes increase expression at 25°C relative to 20°C, indicating elevated metabolism at warm temperature. These data demonstrate that the temperature-induced longevity response is neurally regulated and also provide a partial molecular basis for the rate of living theory, suggesting that these two views are not mutually exclusive. Genetic manipulation and functional assays further uncovered that the NPR-8-dependent longevity response to warm temperature is achieved by regulating the expression of a subset of collagen genes. As increased collagen expression is a common feature of many lifespan-extending interventions and enhanced stress resistance, collagen expression could be critical for healthy aging.

Correction: An epidemiological study of gastrointestinal nematode and Eimeria coccidia infections in different populations of Kazakh sheep.

[This corrects the article DOI: 10.1371/journal.pone.0251307.].

Neuronal GPCR NMUR-1 regulates distinct immune responses to different pathogens.

A key question in current immunology is how the innate immune system generates high levels of specificity. Using the Caenorhabditis elegans model system, we demonstrate that functional loss of NMUR-1, a neuronal G-protein-coupled receptor homologous to mammalian receptors for the neuropeptide neuromedin U, has diverse effects on C. elegans innate immunity against various bacterial pathogens. Transcriptomic analyses and functional assays reveal that NMUR-1 modulates C. elegans transcription activity by regulating the expression of transcription factors involved in binding to RNA polymerase II regulatory regions, which, in turn, controls the expression of distinct immune genes in response to different pathogens. These results uncover a molecular basis for the specificity of C. elegans innate immunity. Given the evolutionary conservation of NMUR-1 signaling in immune regulation across multicellular organisms, our study could provide mechanistic insights into understanding the specificity of innate immunity in other animals, including mammals.

Interactions of human mismatch repair proteins MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway.

At clinically relevant doses, chemotherapeutic S(N)1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSalpha and MutLalpha. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSalpha with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLalpha with TopBP1 and Claspin. We were unable to detect interaction of MutSalpha or MutLalpha with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSalpha with ATR, TopBP1, and Chk1 and of MutLalpha with TopBP1. MutSalpha-Claspin and MutLalpha-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLalpha- and MutSalpha-dependent fashion after N-methyl-N'-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.

Detection of Pathogens and Regulation of Immunity by the Caenorhabditis elegans Nervous System.

Although Caenorhabditis elegans has been used as a model host for studying host-pathogen interactions for more than 20 years, the mechanisms by which it identifies pathogens are not well understood. This is largely due to its lack of most known pattern recognition receptors (PRRs) that recognize pathogen-derived molecules. Recent behavioral research in C. elegans indicates that its nervous system plays a major role in microbe sensing. With the increasing integration of neurobiology in immunological research, future studies may find that neuronal detection of pathogens is an integral part of C. elegans-pathogen interactions. Similar to that of mammals, the C. elegans nervous system regulates its immune system to maintain immunological homeostasis. Studies in the nematode have revealed unprecedented details regarding the molecules, cells, and signaling pathways involved in neural regulation of immunity. Notably, some of the studies indicate that some neuroimmune regulatory circuits need not be "activated" by pathogen infection because they are tonically active and that there could be a predetermined set point for internal immunity, around which the nervous system adjusts immune responses to internal or external environmental changes. Here, we review recent progress on the roles of the C. elegans nervous system in pathogen detection and immune regulation. Because of its advantageous characteristics, we expect that the C. elegans model will be critical for deciphering complex neuroimmune signaling mechanisms that integrate and process multiple sensory cues.

An epidemiological study of gastrointestinal nematode and Eimeria coccidia infections in different populations of Kazakh sheep.

This is an epidemiological study on the gastrointestinal nematode (GIN) and Eimeria coccidia infections in Kazakh sheep and the F1 and F2 generations of Kazakh × Texel sheep crosses. A total of 7599 sheep fecal samples were collected from the Zhaosu County and Nilka County in Ili Kazakh Autonomous Prefecture in the four seasons-spring, summer, autumn, and winter of 2019. The parasite causing the infection was identified by the saturated saline floating method, and the infection intensity was calculated by the modified McMaster method. SPSS19.0 was used to evaluate the differences in the fecal egg count (FEC) of for GIN and the fecal oocyst count (FOC) value of for coccidia per sample. The results showed that there were nine types of sheep GIN infections and Eimeria coccidia in these two counties of Ililocations, with the dominant parasite species of Haemonchus contortus, Trichostrongylus spp., and Ostertagia spp as the predominant parasites in the sheep. Most of the GIN and coccidia infections in these two regions were mild and moderate. The mean log (FEC) of GIN infection in the Zhaosu area was significantly higher than that in the Nilka area, whereas the mean log (FOC) of coccidia infection in Zhaosu was significantly lower than that of Nilka. The mean log (FEC) of GIN infection in the four seasons was the highest in spring, followed by in summer, then in autumn, and the lowest in winter. The mean log (FOC) of coccidia infection was the highest in spring, followed by in autumn, and was the lowest in summer and winter. The mean log (FEC) of GIN infection and log (FOC) of coccidia infection of Kazakh sheep was significantly higher than the F1 generation, which was then significantly higher than the F2 generation of summer. A positive correlation was found between the EPG and OPG levels in the sheep. These results showed that the GIN and coccidia infection intensities of the F1 generation sheep of Kazakh ×Texel crosses were significantly lower than that of Kazakh sheep paving the way for marker-based resistance selection.

NIR-Triggered Blasting Nanovesicles for Targeted Multimodal Image-Guided Synergistic Cancer Photothermal and Chemotherapy.

Escorting therapeutics for malignancies by nano-encapsulation to ameliorate treatment effects and mitigate side effects has been pursued in precision medicine. However, the majority of drug delivery systems suffer from uncontrollable drug release kinetics and thus lead to unsatisfactory triggered-release efficiency along with severe side effects. Herein, we developed a unique nanovesicle delivery system that shows near-infrared (NIR) light-triggered drug release behavior and minimal premature drug release. By co-encapsulation of superparamagnetic iron oxide (SPIO) nanoparticles, the ultrasound contrast agent perfluorohexane (PFH), and cisplatin in a silicate-polyaniline vesicle, we achieved the controllable release of cisplatin in a thermal-responsive manner. Specifically, vaporization of PFH triggered by the heat generated from NIR irradiation imparts high inner vesicle pressure on the nanovesicles, leading to pressure-induced nanovesicle collapse and subsequent cisplatin release. Moreover, the multimodal imaging capability can track tumor engagement of the nanovesicles and assess their therapeutic effects. Due to its precise inherent NIR-triggered drug release, our system shows excellent tumor eradication efficacy and biocompatibility in vivo, empowering it with great prospects for future clinical translation.

A novel in vitro Caenorhabditis elegans transcription system.

BACKGROUND: Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system. RESULTS: By employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner. CONCLUSIONS: In this study, we developed an in vitro C. elegans transcription system that re-constitutes transcription reactions with nuclear extract of larval or adult worms and can both qualitatively and quantitatively detect transcription activity using non-radioactive approaches. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

Altered Brain Regional Homogeneity in First-Degree Relatives of Type 2 Diabetics: A functional MRI Study.

OBJECTIVE: This study aimed to investigate regional homogeneity in the first-: degree relatives of type 2 diabetes patients. METHODS: Seventy-eight subjects, including 26 type 2 diabetes patients, 26 first-: degree relatives, and 26 healthy controls, were assessed. All participants underwent resting-state functional magnetic resonance imaging scanning. The estimated regional homogeneity value was used to evaluate differences in brain activities. RESULTS: In first-: degree relatives, we observed significantly decreased regional homogeneity in the left anterior cingulate cortex, left insula, and bilateral temporal lobes, and increased regional homogeneity in the left superior frontal gyrus, right anterior cingulate cortex, and bilateral posterior cingulate cortex compared to healthy controls. In type 2 diabetes patients, we detected altered regional homogeneity in the left anterior cingulate cortex, left insula, bilateral posterior cingulate cortex, and several other brain regions compared to healthy controls. Both first-: degree relatives and type 2 diabetes patients showed decreased regional homogeneity in the left superior temporal gyrus, right middle temporal gyrus, left anterior cingulate cortex, left insula, and increased regional homogeneity in the left superior frontal gyrus and bilateral posterior cingulate cortex. CONCLUSION: These findings suggest that altered regional homogeneity in the left anterior cingulate cortex, left insula, left superior frontal gyrus, bilateral posterior cingulate cortex, and bilateral temporal lobes might be a neuroimaging biomarker of type 2 diabetes -: related brain dysfunction.