Identification and Characterization of the Glutathione S-Transferase Gene Family in Blueberry (Vaccinium corymbosum) and Their Potential Roles in Anthocyanin Intracellular Transportation.

The glutathione S-transferases (GSTs, EC 2.5.1.18) constitute a versatile enzyme family with pivotal roles in plant stress responses and detoxification processes. Recent discoveries attributed the additional function of facilitating anthocyanin intracellular transportation in plants to GSTs. Our study identified 178 VcGST genes from 12 distinct subfamilies in the blueberry genome. An uneven distribution was observed among these genes across blueberry's chromosomes. Members within the same subfamily displayed homogeneity in gene structure and conserved protein motifs, whereas marked divergence was noted among subfamilies. Functional annotations revealed that VcGSTs were significantly enriched in several gene ontology and KEGG pathway categories. Promoter regions of VcGST genes predominantly contain light-responsive, MYB-binding, and stress-responsive elements. The majority of VcGST genes are subject to purifying selection, with whole-genome duplication or segmental duplication serving as key processes that drive the expansion of the VcGST gene family. Notably, during the ripening of the blueberry fruit, 100 VcGST genes were highly expressed, and the expression patterns of 24 of these genes demonstrated a strong correlation with the dynamic content of fruit anthocyanins. Further analysis identified VcGSTF8, VcGSTF20, and VcGSTF22 as prime candidates of VcGST genes involved in the anthocyanin intracellular transport. This study provides a reference for the exploration of anthocyanin intracellular transport mechanisms and paves the way for investigating the spectrum of GST functions in blueberries.

Positive effect of microvascular proliferation on functional recovery in experimental cervical spondylotic myelopathy.

Background and purpose: Cervical Spondylotic Myelopathy (CSM), the most common cause of spinal cord dysfunction globally, is a degenerative disease that results in non-violent, gradual, and long-lasting compression of the cervical spinal cord. The objective of this study was to investigate whether microvascular proliferation could positively affect neural function recovery in experimental cervical spondylotic myelopathy (CSM). Methods: A total of 60 male adult Sprague-Dawley (SD) were randomly divided into four groups: Control (CON), Compression (COM), Angiostasis (AS), and Angiogenesis (A G),with 15 rats in each group. Rats in the AS group received SU5416 to inhibit angiogenesis, while rats in the AG group received Deferoxamine (DFO) to promote angiogenesis. Motor and sensory functions were assessed using the Basso Beattie Bresnahan (BBB) scale and somatosensory evoked potential (SEP) examination. Neuropathological degeneration was evaluated by the number of neurons, Nissl bodies (NB), and the de-myelination of white matter detected by Hematoxylin & Eosin(HE), Toluidine Blue (TB), and Luxol Fast Blue (LFB) staining. Immunohistochemical (IHC) staining was used to observe the Neurovascular Unit (NVU). Results: Rats in the CON group exhibited normal locomotor function with full BBB score, normal SEP latency and amplitude. Among the other three groups, the AG group had the highest BBB score and the shortest SEP latency, while the AS group had the lowest BBB score and the most prolonged SEP latency. The SEP amplitude showed an opposite performance to the latency. Compared to the COM and AS groups, the AG group demonstrated significant neuronal restoration in gray matter and axonal remyelination in white matter. DFO promoted microvascular proliferation, especially in gray matter, and improved the survival of neuroglial cells. In contrast, SU-5416 inhibited the viability of neuroglial cells by reducing micro vessels. Conclusion: The microvascular status was closely related to NVU remodeling an-d functional recovery. Therefore, proliferation of micro vessels contributed to function -al recovery in experimental CSM, which may be associated with NVU remodeling.

Osteoclast Cancer Cell Metabolic Cross-talk Confers PARP Inhibitor Resistance in Bone Metastatic Breast Cancer.

The majority of patients with late-stage breast cancer develop distal bone metastases. The bone microenvironment can affect response to therapy, and uncovering the underlying mechanisms could help identify improved strategies for treating bone metastatic breast cancer. Here, we observed that osteoclasts reduced the sensitivity of breast cancer cells to DNA damaging agents, including cisplatin and the PARP inhibitor (PARPi) olaparib. Metabolic profiling identified elevated glutamine production by osteoclasts. Glutamine supplementation enhanced the survival of breast cancer cells treated with DNA damaging agents, while blocking glutamine uptake increased sensitivity and suppressed bone metastasis. GPX4, the critical enzyme responsible for glutathione oxidation, was upregulated in cancer cells following PARPi treatment through stress-induced ATF4-dependent transcriptional programming. Increased glutamine uptake and GPX4 upregulation concertedly enhanced glutathione metabolism in cancer cells to help neutralize oxidative stress and generate PARPi resistance. Analysis of paired patient samples of primary breast tumors and bone metastases revealed significant induction of GPX4 in bone metastases. Combination therapy utilizing PARPi and zoledronate, which blocks osteoclast activity and thereby reduces the microenvironmental glutamine supply, generated a synergistic effect in reducing bone metastasis. These results identify a role for glutamine production by bone-resident cells in supporting metastatic cancer cells to overcome oxidative stress and develop resistance to DNA-damaging therapies. SIGNIFICANCE: Metabolic interaction between osteoclasts and tumor cells contributes to resistance to DNA-damaging agents, which can be blocked by combination treatment with PARP and osteoclast inhibitors to reduce bone metastatic burden.

Characterization and Assessment of Sheep-Origin Probiotic Bacillus licheniformis B63 Strain for Potential Use in Intestinal Health and Disease.

Bacterial diarrhea causes serious losses for the sheep industry. Antibiotic resistance acquired by diarrheal bacteria is still a hurdle in the care of animal health. Thus, it is urgent to develop effective alternatives to antibiotics for controlling bacterial diarrhea. We initially isolated Bacillus spp. from Xinjiang fine wool sheep fecal and determined their properties of hemolysis and tolerance to acid and bile salts to identify potential candidates. Subsequently, we studied the position of a candidate in phylogenetic trees by 16S rRNA sequences and its susceptibility to antibiotics, ability to inhibit diarrheal bacteria, and toxicity, as well as its effects on animal health. Fourteen Bacillus spp. strains were isolated from sheep fecal. We identified the non-hemolysis B63 strain, which exhibited a high tolerance to acid and bile salts. Phylogenetic analysis indicated that the B63 strain is a new strain of Bacillus licheniformis. The B. licheniformis B63 strain was prompt to form spores, susceptible to commonly used antibiotics, and able to inhibit diarrhea-associated bacteria, including Escherichia coli, Staphylococcus aureus, and Salmonella typhi. Animal studies determined that B. licheniformis B63 at 4 × 108 CFU/mL was non-toxic to mice and SD rats. Supplement with B. licheniformis B63 promoted the body weight gain of mice, reduced the inflammatory interleukin 6, and increased the jejunum villus height of SD rats. The newly isolated, non-hemolysis, spore-forming B. licheniformis B63 strain should be considered an optimal strain for the development of an effective probiotic supplement to control diarrheal diseases and promote the health of sheep and other animals.

Genome-Wide Identification and Analysis of the MADS-Box Transcription Factor Genes in Blueberry (Vaccinium spp.) and Their Expression Pattern during Fruit Ripening.

MADS-box is a class of transcriptional regulators that are ubiquitous in plants and plays important roles in the process of plant growth and development. Identification and analysis of blueberry MADS-box genes can lay a foundation for their function investigations. In the present study, 249 putative MADS-box genes were identified in the blueberry genome. Those MADS-box genes were distributed on 47 out of 48 chromosomes. The phylogenetic and evolutionary analyses showed that blueberry MADS-box genes were divided into 131 type I members and 118 type II members. The type I genes contained an average of 1.89 exons and the type II genes contained an average of 7.83 exons. Motif analysis identified 15 conserved motifs, of which 4 were related to the MADS domain and 3 were related to the K-box domain. A variety of cis-acting elements were found in the promoter region of the blueberry MADS-box gene, indicating that the MADS-box gene responded to various hormones and environmental alterations. A total of 243 collinear gene pairs were identified, most of which had a Ka/Ks value of less than 1. Nine genes belonging to SEP, AP3/PI, and AGL6 subfamilies were screened based on transcriptomic data. The expression patterns of those nine genes were also verified using quantitative PCR, suggesting that VcMADS6, VcMADS35, VcMADS44, VcMADS58, VcMADS125, VcMADS188, and VcMADS212 had potential functions in blueberry fruit ripening. The results of this study provide references for an in-depth understanding of the biological function of the blueberry MADS-box genes and the mechanism of blueberry fruit ripening.

Deep Learning-Based Auto-Segmentation of Spinal Cord Internal Structure of Diffusion Tensor Imaging in Cervical Spondylotic Myelopathy.

Cervical spondylotic myelopathy (CSM) is a chronic disorder of the spinal cord. ROI-based features on diffusion tensor imaging (DTI) provide additional information about spinal cord status, which would benefit the diagnosis and prognosis of CSM. However, the manual extraction of the DTI-related features on multiple ROIs is time-consuming and laborious. In total, 1159 slices at cervical levels from 89 CSM patients were analyzed, and corresponding fractional anisotropy (FA) maps were calculated. Eight ROIs were drawn, covering both sides of lateral, dorsal, ventral, and gray matter. The UNet model was trained with the proposed heatmap distance loss for auto-segmentation. Mean Dice coefficients on the test dataset for dorsal, lateral, and ventral column and gray matter were 0.69, 0.67, 0.57, 0.54 on the left side and 0.68, 0.67, 0.59, 0.55 on the right side. The ROI-based mean FA value based on segmentation model strongly correlated with the value based on manual drawing. The percentages of the mean absolute error between the two values of multiple ROIs were 0.07, 0.07, 0.11, and 0.08 on the left side and 0.07, 0.1, 0.1, 0.11, and 0.07 on the right side. The proposed segmentation model has the potential to offer a more detailed spinal cord segmentation and would be beneficial for quantifying a more detailed status of the cervical spinal cord.

Neurovascular Unit Compensation from Adjacent Level May Contribute to Spontaneous Functional Recovery in Experimental Cervical Spondylotic Myelopathy.

The progression and remission of cervical spondylotic myelopathy (CSM) are quite unpredictable due to the ambiguous pathomechanisms. Spontaneous functional recovery (SFR) has been commonly implicated in the natural course of incomplete acute spinal cord injury (SCI), while the evidence and underlying pathomechanisms of neurovascular unit (NVU) compensation involved in SFR remains poorly understood in CSM. In this study, we investigate whether compensatory change of NVU, in particular in the adjacent level of the compressive epicenter, is involved in the natural course of SFR, using an established experimental CSM model. Chronic compression was created by an expandable water-absorbing polyurethane polymer at C5 level. Neurological function was dynamically assessed by BBB scoring and somatosensory evoked potential (SEP) up to 2 months. (Ultra)pathological features of NVUs were presented by histopathological and TEM examination. Quantitative analysis of regional vascular profile area/number (RVPA/RVPN) and neuroglial cells numbers were based on the specific EBA immunoreactivity and neuroglial biomarkers, respectively. Functional integrity of blood spinal cord barrier (BSCB) was detected by Evan blue extravasation test. Although destruction of the NVU, including disruption of the BSCB, neuronal degeneration and axon demyelination, as well as dramatic neuroglia reaction, were found in the compressive epicenter and spontaneous locomotor and sensory function recovery were verified in the modeling rats. In particular, restoration of BSCB permeability and an evident increase in RVPA with wrapping proliferated astrocytic endfeet in gray matter and neuron survival and synaptic plasticity were confirmed in the adjacent level. TEM findings also proved ultrastructural restoration of the NVU. Thus, NVU compensation changes in the adjacent level may be one of the essential pathomechanisms of SFR in CSM, which could be a promising endogenous target for neurorestoration.

Ultrastructural destruction of neurovascular unit in experimental cervical spondylotic myelopathy.

Background and purpose: The pathogenesis of cervical spondylotic myelopathy (CSM) remains unclear. This study aimed to explore the ultrastructural pathology of neurovascular unit (NVU) during natural development of CSM. Methods: A total of 24 rats were randomly allocated to the control group and the CSM group. Basso-Beattie-Bresnahan (BBB) scoring and somatosensory evoked potentials (SEP) were used as functional assessments. Hematoxylin-eosin (HE), toluidine blue (TB), and Luxol fast blue (LFB) stains were used for general structure observation. Transmission electron microscopy (TEM) was applied for investigating ultrastructural characteristics. Results: The evident compression caused significant neurological dysfunction, which was confirmed by the decrease in BBB score and SEP amplitude, as well as the prolongation of SEP latency (P < 0.05). The histopathological findings verified a significant decrease in the amount of Nissl body and myelin area and an increase in vacuolation compared with the control group (P < 0.05). The TEM results revealed ultrastructural destruction of NVU in several forms, including: neuronal degeneration and apoptosis; disruption of axonal cytoskeleton (neurofilaments) and myelin sheath and dystrophy of axonal terminal with dysfunction mitochondria; degenerative oligodendrocyte, astrocyte, and microglial cell inclusions with degenerating axon and dystrophic dendrite; swollen microvascular endothelium and loss of tight junction integrity; corroded basement membrane and collapsed microvascular wall; and proliferated pericyte and perivascular astrocytic endfeet. In the CSM group, reduction was observed in the amount of mitochondria with normal appearance and the number of cristae per mitochondria (P < 0.05), while no substantial drop of synaptic vesicle number was seen (P > 0.05). Significant narrowing of microvascular lumen size was also observed, accompanied by growth in the vascular wall area, endothelial area, basement membrane thickness, astrocytic endfeet area, and pericyte coverage area (rate) (P < 0.05). Conclusion: Altogether, the findings of this study demonstrated ultrastructural destruction of NVU in an experimental CSM model with dorsal-lateral compression, revealing one of the crucial pathophysiological mechanisms of CSM.

Colicins of Escherichia coli Lead to Resistance against the Diarrhea-Causing Pathogen Enterotoxigenic E. coli in Pigs.

Gut microbes can affect host adaptation to various environment conditions. Escherichia coli is a common gut species, including pathogenic strains and nonpathogenic strains. This study was conducted to investigate the effects of different E. coli strains in the gut on the health of pigs. In this study, the complete genomes of two E. coli strains isolated from pigs were sequenced. The whole genomes of Y18J and the enterotoxigenic E. coli strain W25K were compared to determine their roles in pig adaptation to disease. Y18J was isolated from feces of healthy piglets and showed strong antimicrobial activity against W25K in vitro. Gene knockout experiments and complementation analysis followed by modeling the microbe-microbe interactions demonstrated that the antagonistic mechanism of Y18J against W25K relied on the bacteriocins colicin B and colicin M. Compared to W25K, Y18J is devoid of exotoxin-coding genes and has more secondary-metabolite-biosynthetic gene clusters. W25K carries more genes involved in genome replication, in accordance with a shorter cell cycle observed during a growth experiment. The analysis of gut metagenomes in different pig breeds showed that colicins B and M were enriched in Laiwu pigs, a Chinese local breed, but were scarce in boars and Duroc pigs. IMPORTANCE This study revealed the heterogeneity of E. coli strains from pigs, including two strains studied by both in silico and wet experiments in detail and 14 strains studied by bioinformatics analysis. E. coli Y18J may improve the adaptability of pigs toward disease resistance through the production of colicins B and M. Our findings could shed light on the pathogenic and harmless roles of E. coli in modern animal husbandry, leading to a better understanding of intestinal-microbe-pathogen interactions in the course of evolution.

Flexible Bronchoscope versus Video Laryngoscope for Orotracheal Intubation During Upper Gastrointestinal Endoscopic Surgery in Left Lateral Position: A Randomized Controlled Trial.

Purpose: Endoscopic submucosal dissection (ESD) has become the primary treatment for early upper gastrointestinal tract lesions. During endoscopic surgery, endotracheal intubation is generally performed in the patients' supine position, and patients are shifted to the left lateral position for endoscopic surgery. This study compared the efficacy of flexible bronchoscope-guided intubation with that of video laryngoscope-guided intubation in the left lateral position. Patients and Methods: Forty-eight patients receiving ESD were randomly divided into the flexible bronchoscope group (group F) or the video laryngoscope group (group V). Tracheal intubation was performed by a trained anesthetist with a flexible bronchoscope (group F) or unchanneled video laryngoscope (group V) in the left lateral position. Primary outcomes included the intubation duration and success rate. Secondary outcomes included the ease of intubation technique and the occurrence of complications. Results: Twenty-four (100%) patients in group F and twenty-three (95.8%) in group V were successfully intubated (P = 1.000). The median intubation time in group F was 37s (interquartile range, 33.0, 44.5), which was significantly shorter compared to group V (53s [45.5, 66.5]; P < 0.001). The flexible bronchoscope was significantly easier to manage than the video laryngoscope, as reflected by the users scoring system (9 [9, 10] vs 8 [7, 8]; P < 0.001). The presence of perioperative adverse events and complications were comparable between the two groups. Conclusion: Both flexible bronchoscope- and video laryngoscope-guided intubation in patients' left lateral position achieved high success rates and comparable complication rates. However, intubation with the flexible bronchoscope was completed more quickly.

Tumor-derived Jagged1 promotes cancer progression through immune evasion.

Immune checkpoint inhibitor (ICI) therapy is generating remarkable responses in individuals with cancer, but only a small portion of individuals with breast cancer respond well. Here we report that tumor-derived Jagged1 is a key regulator of the tumor immune microenvironment. Jagged1 promotes tumorigenesis in multiple spontaneous mammary tumor models. Through Jagged1-induced Notch activation, tumor cells increase expression and secretion of multiple cytokines to help recruit macrophages into the tumor microenvironment. Educated macrophages crosstalk with tumor-infiltrating T cells to inhibit T cell proliferation and tumoricidal activity. In individuals with triple-negative breast cancer, a high expression level of Jagged1 correlates with increased macrophage infiltration and decreased T cell activity. Co-administration of an ICI PD-1 antibody with a Notch inhibitor significantly inhibits tumor growth in breast cancer models. Our findings establish a distinct signaling cascade by which Jagged1 promotes adaptive immune evasion of tumor cells and provide several possible therapeutic targets.

ASB13 inhibits breast cancer metastasis through promoting SNAI2 degradation and relieving its transcriptional repression of YAP.

Transcription factor SNAI2 plays key roles during development and has also been known to promote metastasis by inducing invasive phenotype and tumor-initiating activity of cancer cells. However, the post-translational regulation of SNAI2 is less well studied. We performed a dual-luciferase-based, genome-wide E3 ligase siRNA library screen and identified ASB13 as an E3 ubiquitin ligase that targets SNAI2 for ubiquitination and degradation. ASB13 knockout in breast cancer cells promoted cell migration and decreased F-actin polymerization, while overexpression of ASB13 suppressed lung metastasis. Furthermore, ASB13 knockout decreased YAP expression, and such regulation is dependent on an increased protein level of SNAI2, which in turn represses YAP transcription. YAP suppresses tumor progression in breast cancer, as YAP knockout increases tumorsphere formation, anchorage-independent colony formation, cell migration in vitro, and lung metastasis in vivo. Clinical data analysis reveals that ASB13 expression is positively correlated with improved overall survival in breast cancer patients. These findings establish ASB13 as a suppressor of breast cancer metastasis by promoting degradation of SNAI2 and relieving its transcriptional repression of YAP.

Surface-Enhanced Infrared Absorption of Ligands on Colloidal Gold Nanowires through Resonant Coupling.

Pushing the detection limit of infrared absorption (IR) through surface-enhanced (SEIRA) approaches have far-reaching prospect for related applications in molecular analysis and detection. Specifically engineered Au nanowires (NWs) can be applied as the surface-enhancing substrates in colloidal solution, given their longitudinal surface plasmon resonance (SPR) being aspect-ratio dependent and extendable into the infrared region. Through carefully designed control experiments, we realized resonant coupling between the longitudinal surface plasmons of Au NWs and the vibration modes of the bonded oleylamine (OA) ligands. In our system, after deliberately tuning thickness of the OA ligands and ratio of the detached/attached ligands in the solution, the apparent enhancement factor of IR signal from ligands around Au NWs could be pushed up to 5.29 × 104. Given the facile tuning of SPR properties of Au NWs in the colloidal solution and the performance demonstrated in the report, our work could be an intriguing platform for SEIRA implementations in a broad spectrum of circumstances.

Reliability and Validity of the Chinese Version of Modified Checklist for Autism in Toddlers, Revised, with Follow-Up (M-CHAT-R/F).

Although early detection of autism facilitates intervention, early detection strategies are not yet widespread in China. To improve the situation, the Chinese version of the Modified Checklist for Autism in Toddlers, Revised with Follow-Up (M-CHAT-R/F) was validated. The sample included 7928 toddlers, aged 16 to 30 months, screened during their routine care in six provinces of China. When the cut-off value was 3, the sensitivity and specificity of M-CHAT-R were 0.963 and 0.865. The inter-rater reliability and the test-retest reliability were also adequate (intraclass correlation coefficients were 0.853 and 0.759, both ps < .01). The Chinese version of M-CHAT-R/F is an effective tool for early detection of ASD and is applicable to early screening in China.

Effects of neoadjuvant chemotherapy on the depth of total intravenous anesthesia in patients with breast cancer undergoing unilateral modified radical mastectomy: A prospective observational study.

Toxic effects of neoadjuvant chemotherapy (NC) on nervous, hepatorenal, and pulmonary systems might affect general anesthesia depth. This study aimed to evaluate the effects of NC on depth of total intravenous anesthesia.This prospective observational study enrolled 60 patients undergoing elective unilateral modified radical mastectomy during total intravenous anesthesia with propofol and remifentanil (January-June 2015; Liaocheng People's Hospital, China): the NC group (n = 30) received NC, while the control group (n = 30) did not. Propofol and remifentanil dosages were adjusted according to indexes of consciousness (IoC1: sedation; IoC2: analgesia) to control fluctuations of blood pressure and heart rate within 20% of baseline values. Parameters reflecting propofol/remifentanil dosages, intraoperative adverse events, and quality of anesthetic recovery were recorded.The duration of propofol infusion (1.3 ±â€Š0.4 vs 1.8 ±â€Š0.5 hours, P < .05), mean propofol dosage (8.0 ±â€Š1.0 vs 9.3 ±â€Š1.5 mg kg h, P < .05), and adjustment frequency of target-controlled remifentanil infusion (2.9 ±â€Š1.8 vs 4.4 ±â€Š2.6 times/surgery, P < .05) were significantly lower in the NC group than in the control group; adjustment frequency of target-controlled propofol infusion was also numerically lower (2.0 ±â€Š1.1 vs 2.7 ±â€Š1.5 times/surgery, P = .053). Duration of remifentanil infusion, mean remifentanil dosage, voluntary eye opening, extubation time, and recovery score were not significantly different between groups. The incidence of tachycardia was lower in the NC group than in the control group (7.1% vs 37.0%, P < .05), but there was no significant difference in the incidence of total adverse events between groups.NC can enhance the sensitivity of breast cancer patients to the anesthetic effect of propofol.

Effects of indexes of consciousness (IoC1 and IoC2) monitoring on remifentanil dosage in modified radical mastectomy: a randomized trial.

BACKGROUND: This study investigated the effects of indexes of consciousness (IoC1 and IoC2) monitoring on remifentanil dosage. METHODS: In this randomized, single-blinded, prospective study, 120 patients undergoing unilateral modified radical mastectomy were randomly assigned to the treatment group (T group, n = 60) or control group (C group, n = 60). In the T group, patients received both IoC1 (sedation) and IoC2 (analgesia) monitoring, and remifentanil dosages were adjusted by anesthetists according to IoC2. In the C group, remifentanil dosages were adjusted based on the anesthetists' judgment according to the patients' vital signs. Remifentanil dose, adjustment frequency, infusion duration, intraoperative adverse events, and quality of anesthetic recovery were compared between the two groups. The primary outcome was the dose of remifentanil. RESULTS: Compared with the C group, mean remifentanil dosage was significantly higher in the T group (3.8 ± 1.9 versus 3.2 ± 1.2 µg kg(-1) h(-1), P < 0.05) during the anesthetic period, as was the adjustment frequency of target-controlled infusion (2.9 ± 1.9 versus 2.0 ± 1.2 times/surgery, P < 0.05), but there was no difference in infusion duration. Voluntary eye opening, extubation time, and recovery score were not significantly different between the two groups (P > 0.05). Total adverse events were significantly reduced in the T group (P < 0.05). CONCLUSIONS: IoC1-targeted propofol dosing does not seem to be significantly different to hemodynamic-based monitoring, whereas IoC2 monitoring can increase remifentanil dosage during modified radical mastectomy, but the anesthetic process is more controllable and total adverse events are reduced, which improves the controllability of anesthesia. TRIAL REGISTRATION NUMBER: ChiCTR-TRC-13004101 , registered on 27 November 2013.

Ligase I and ligase III mediate the DNA double-strand break ligation in alternative end-joining.

In eukaryotes, DNA double-strand breaks (DSBs), one of the most harmful types of DNA damage, are repaired by homologous repair (HR) and nonhomologous end-joining (NHEJ). Surprisingly, in cells deficient for core classic NHEJ factors such as DNA ligase IV (Lig4), substantial end-joining activities have been observed in various situations, suggesting the existence of alternative end-joining (A-EJ) activities. Several putative A-EJ factors have been proposed, although results are mostly controversial. By using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, we generated mouse CH12F3 cell lines in which, in addition to Lig4, either Lig1 or nuclear Lig3, representing the cells containing a single DNA ligase (Lig3 or Lig1, respectively) in their nucleus, was completely ablated. Surprisingly, we found that both Lig1- and Lig3-containing complexes could efficiently catalyze A-EJ for class switching recombination (CSR) in the IgH locus and chromosomal deletions between DSBs generated by CRISPR/Cas9 in cis-chromosomes. However, only deletion of nuclear Lig3, but not Lig1, could significantly reduce the interchromosomal translocations in Lig4(-/-) cells, suggesting the unique role of Lig3 in catalyzing chromosome translocation. Additional sequence analysis of chromosome translocation junction microhomology revealed the specificity of different ligase-containing complexes. The data suggested the existence of multiple DNA ligase-containing complexes in A-EJ.

Enzymatic synthesis of modified oligonucleotides by PEAR using Phusion and KOD DNA polymerases.

Antisense synthetic oligonucleotides have been developed as potential gene-targeted therapeutics. We previously reported polymerase-endonuclease amplification reaction (PEAR) for amplification of natural and 5'-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2'-deoxy-2'-fluoro-(2'-F) and 2'-F/S double-modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with one or two modified nucleotides, and KOD DNA polymerase is more suitable than Phusion DNA polymerase for PEAR amplification of 2'-F and 2'-F/S double modified oligonucleotides. The composition of PEAR products were analyzed by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection and showed that the sequence of the PEAR products are maintained at an extremely high accuracy (>99.9%), and after digestion the area percent of full-length modified oligonucleotides reaches 89.24%. PEAR is suitable for synthesis of modified oligonucleotides efficiently and with high purity.

High-sensitivity naphthalene-based two-photon fluorescent probe suitable for direct bioimaging of H2S in living cells.

H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S in living systems are desired. Although quite a few one photon fluorescence probes have been reported for H2S, two-photon (TP) probes are more favorable for intracellular imaging. In this work, by employing a donor-π-acceptor-structured naphthalene derivative as the two-photon fluorophore and an azide group as the recognition unit, we reported a new two-photon bioimaging probe 6-(benzo[d]thiazol-2'-yl)-2-azidonaphthalene (NHS1) for H2S with improved sensitivity. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for both one photon and TP excited fluorescence were observed, resulting in a high sensitivity to H2S in aqueous solutions with a detection limit of 20 nM observed, much lower than the previously reported TP probe. The probe also exhibits a wide linear response concentration range (0-5 µM) to H2S with high selectivity. All these features are favorable for direct monitoring of H2S in complex biological samples. It was then applied for direct TP imaging of H2S in living cells with satisfactory sensitivity, demonstrating its value of practical application in biological systems.

Development and applications of nano-powder patents--a review in patents.

A thorough review on the patents in the applications of nano-powder technology shows that research work in this technical field is growing stronger, as indicated by a rapid increase in the number of independent patents in relevant topics. This also indicates ever increasing and continuous interests on the nano-powder technology even after 15 years of dynamic global nanotechnology development. The review on the patents in the nano-powder area shows that the use of specialized databanks is recommendable and beneficial in providing the researchers in this technical community an updated, systematic and rapid reference in furthering new development and expansion of this vital nanotechnology field.