Isolation and characterization of oxidizedoligogalacturonides: meccanism of dampening of damps

Oligogalacturonides (OGs) released upon partial degradation of homogalacturonan, are a well-known class of Damage-Associated Molecular Patterns (DAMPs). Besides inducing immunity, OGs negatively affect plant growth by antagonizing auxin responses. Because the recognition of DAMPs poses the intrinsic risk of activating an exaggerated response that may impair plant survival, dampening mechanisms of DAMPs should exist. Transgenic Arabidopsis plants (OGM plants) expressing a chimeric protein called "OGmachine" accumulate oligogalacturonides (OGs) in their tissues and exhibit enhanced resistance to a variety of pathogens; however the growth of these plants is severely impaired. The prolonged release of OGs triggers defense responses that in the long term are deleterious for the plant. We used the OGM plants as a tool to investigate a possible regulatory mechanism by searching for elicitor-inactive OGs that may derive from elicitor-active OGs through an enzymatic modification. By analyzing the OGs produced in the transgenic plants, modified OGs were isolated. The nature of the modification was investigated by electrospray ionization mass spectrometry and resulted to be the oxidation to galactaric acid of the residue at the reducing end of OGs (oxOGs). OxOGs were tested for their ability to induce defense responses and antagonize auxin responses. In all experiments, they were inactive as compared to the corresponding typical OGs. We succeeded to isolate and characterize one of the enzymes that causes the inactivation of OGs: it is a FAD binding oxidase, that we named OGOX1, capable of producing elicitor-inactive oxidized OGs and H2O2

Continuous Glucose Monitoring Metrics for Earlier Identification of Pre-Diabetes: Protocol for a Systematic Review and Meta-Analysis

Introduction: Glycaemic variability and other metrics are not well characterised in subjects without diabetes. More comprehensive sampling as obtained with continuous glucose monitoring (CGM) may improve diagnostic accuracy of the transition from health to pre-diabetes. Our goal is to investigate the glycaemic system as it shifts from health to pre-disease in adult patients without diabetes using CGM metrics. New insights may offer therapeutic promise for reversing dysglycaemia more successfully with dietary, nutritional and lifestyle change before progression occurs to pre-diabetes and diabetes. Methods and analysis: This systematic review will include comprehensive searches of the PubMed, Scopus, Cochrane Library and ClinicalTrials.gov databases, with restrictions set to studies published in the last 10 years in English and planned search date 10 March 2022. Reference lists of studies that meet eligibility criteria in the screening process will subsequently be screened for the potential inclusion of additional studies. We will include studies that examine CGM use and report diagnostic criteria such as fasting glucose and/or haemoglobin A1c such that we can assess correlation between CGM metrics and established diagnostic criteria and describe how CGM metrics are altered in the transition from health to pre-diabetes. The screening and data extraction will be conducted by two independent reviewers using Covidence. All included papers will also be evaluated for quality and publication bias using Cochrane Collaboration risk of bias tools. If there are two or more studies with quantitative estimates that can be combined, we will conduct a meta-analysis after assessing heterogeneity. Ethics and dissemination: The systematic review methodology does not require formal ethical review due to the nature of the study design. Study findings will be publicly available and published in a peer-reviewed journal. Prospero registration number: CRD42022308222

A Cross-Sectional Study Evaluating the Impact of One Year versus Two Years of Exposure to Interprofessional Education on Student Perceptions of Physician-Pharmacist Interprofessional Clinical Education (SPICE)

Background: Evaluating student perceptions of interprofessional education (IPE) is important to meet accreditation standards. The objective of this study was to evaluate the impact of one year versus two years of exposure to IPE on student perceptions, as well as evaluate differences between professions. Methods: In this cross-sectional study, first and second year medical and pharmacy students enrolled in an interprofessional experiential course series at each of their respective institutions completed a perceptions instrument prior to a standardized objective behavioral assessment. Student demographics and perception scores were summarized using descriptive statistics. Chi-squared tests and Cochran-Mantel-Haenszel tests were used to assess differences in demographic variables. Between-group differences in perception scores were assessed using Wilcoxon Rank-Sum tests. Results: 155 students completed the instrument out of the 163 students enrolled in the course series. Overall, the median scores were ≥4 (4=Agree, 5=Strongly Agree) for all SPICE items and factors. No significant differences were observed between first and second year students in response to any of the SPICE items or factors. When comparing professions, significant differences were observed between pharmacy students and medical students that IPE “enhances my education” (p=0.003), “improves patient satisfaction” (p=0.001), and “enhances my future ability to collaborate” (p=0.001). Significant differences were also observed between pharmacy students and medical students for 2 of the 3 factors: teamwork (p=0.001) and patient outcomes (p=0.005). For all of the differences in items and factors, pharmacy students reported higher levels of agreement. Conclusions: Two years of exposure to IPE compared to one year (i.e. second year students vs. first year students) did not result in higher levels of agreement; however, agreement was high across all students which may have limited the ability to detect a difference. When perceptions are high early in the curriculum, maintaining the same level of agreement longitudinally may be a more appropriate educational outcome. Pharmacy students had higher levels of agreement compared to medical students for certain items. Further research is needed to determine if these differences have an impact on interprofessional collaboration. Conflict of Interest We declare no conflicts of interest or financial interests that the authors or members of their immediate families have in any product or service discussed in the manuscript, including grants (pending or received), employment, gifts, stock holdings or options, honoraria, consultancies, expert testimony, patents and royalties.   Type: Original Researc

A Cross-Sectional Study Evaluating the Impact of One Year versus Two Years of Exposure to Interprofessional Education on Student Perceptions of Physician-Pharmacist Interprofessional Clinical Education (SPICE)

Background: Evaluating student perceptions of interprofessional education (IPE) is important to meet accreditation standards. The objective of this study was to evaluate the impact of one year versus two years of exposure to IPE on student perceptions, as well as evaluate differences between professions. Methods: In this cross-sectional study, first and second year medical and pharmacy students enrolled in an interprofessional experiential course series at each of their respective institutions completed a perceptions instrument prior to a standardized objective behavioral assessment. Student demographics and perception scores were summarized using descriptive statistics. Chi-squared tests and Cochran-Mantel-Haenszel tests were used to assess differences in demographic variables. Between-group differences in perception scores were assessed using Wilcoxon Rank-Sum tests. Results: 155 students completed the instrument out of the 163 students enrolled in the course series. Overall, the median scores were ≥4 (4=Agree, 5=Strongly Agree) for all SPICE items and factors. No significant differences were observed between first and second year students in response to any of the SPICE items or factors. When comparing professions, significant differences were observed between pharmacy students and medical students that IPE “enhances my education” (p=0.003), “improves patient satisfaction” (p=0.001), and “enhances my future ability to collaborate” (p=0.001). Significant differences were also observed between pharmacy students and medical students for 2 of the 3 factors: teamwork (p=0.001) and patient outcomes (p=0.005). For all of the differences in items and factors, pharmacy students reported higher levels of agreement. Conclusions: Two years of exposure to IPE compared to one year (i.e. second year students vs. first year students) did not result in higher levels of agreement; however, agreement was high across all students which may have limited the ability to detect a difference. When perceptions are high early in the curriculum, maintaining the same level of agreement longitudinally may be a more appropriate educational outcome. Pharmacy students had higher levels of agreement compared to medical students for certain items. Further research is needed to determine if these differences have an impact on interprofessional collaboration. Conflict of Interest We declare no conflicts of interest or financial interests that the authors or members of their immediate families have in any product or service discussed in the manuscript, including grants (pending or received), employment, gifts, stock holdings or options, honoraria, consultancies, expert testimony, patents and royalties.   Type: Original Researc

Pregnancy complications in acquired thrombotic thrombocytopenic purpura : a case-control study

BackgroundPregnant women with a history of acquired thrombotic thrombocytopenic purpura (TTP) are considered at risk for disease recurrence and might be at risk for miscarriage, similar to other autoimmune disorders. However, the exact entity of these risks and their causes are unknown. The aim of this study was to evaluate risk factors associated with adverse pregnancy outcome, in terms of both gravidic TTP and miscarriage, in women affected by previous acquired TTP.MethodsWe conducted a nested case\ubfcontrol study in women with a history of acquired TTP enrolled in the Milan TTP registry from 1994 to October 2012, with strict inclusion criteria to reduce referral and selection bias.ResultsFifteen out of 254 women with acquired TTP were included, namely four cases with gravidic TTP, five with miscarriage, and six controls with uncomplicated pregnancy. In the cases, ADAMTS13 activity levels in the first trimester were moderately-to-severely reduced (median levels <3% in gravidic TTP and median levels 20% [range 14-40%] in the women with miscarriage) and anti-ADAMTS13 antibodies were invariably present, while in the control group ADAMTS13 activity levels were normal (median 90%, range 40-129%), with absence of detectable anti-ADAMTS13 antibodies. Reduced levels of ADAMTS13 activity (<25%) in the first trimester were associated with an over 2.9-fold increased risk for gravidic TTP and with an over 1.2-fold increased risk for miscarriage (lower boundary of the confidence interval of the odds ratio). In addition, the presence of anti-ADAMTS13 antibodies during pregnancy was associated with an over 6.6-fold increased risk for gravidic TTP and with an over 4.1-fold increased risk for miscarriage.ConclusionsADAMTS13 activity evaluation and detection of anti-ADAMTS13 antibody could help to predict the risk of complications in pregnant women with a history of acquired TTP

The Dynamical Mechanism of Auto-Inhibition of AMP-Activated Protein Kinase

We use a novel normal mode analysis of an elastic network model drawn from configurations generated during microsecond all-atom molecular dynamics simulations to analyze the mechanism of auto-inhibition of AMP-activated protein kinase (AMPK). A recent X-ray and mutagenesis experiment (Chen, et al Nature 2009, 459, 1146) of the AMPK homolog S. Pombe sucrose non-fermenting 1 (SNF1) has proposed a new conformational switch model involving the movement of the kinase domain (KD) between an inactive unphosphorylated open state and an active or semi-active phosphorylated closed state, mediated by the autoinhibitory domain (AID), and a similar mutagenesis study showed that rat AMPK has the same auto-inhibition mechanism. However, there is no direct dynamical evidence to support this model and it is not clear whether other functionally important local structural components are equally inhibited. By using the same SNF1 KD-AID fragment as that used in experiment, we show that AID inhibits the catalytic function by restraining the KD into an unproductive open conformation, thereby limiting local structural rearrangements, while mutations that disrupt the interactions between the KD and AID allow for both the local structural rearrangement and global interlobe conformational transition. Our calculations further show that the AID also greatly impacts the structuring and mobility of the activation loop

Rebuild, restore, reinnervate: do human tissue engineered dermo-epidermal skin analogs attract host nerve fibers for innervation?

PURPOSE: Tissue engineered skin substitutes are a promising tool to cover large skin defects, but little is known about reinnervation of transplants. In this experimental study, we analyzed the ingrowth of host peripheral nerve fibers into human tissue engineered dermo-epidermal skin substitutes in a rat model. Using varying cell types in the epidermal compartment, we wanted to assess the influence of epidermal cell types on reinnervation of the substitute. METHODS: We isolated keratinocytes, melanocytes, fibroblasts, and eccrine sweat gland cells from human skin biopsies. After expansion, epidermal cells were seeded on human dermal fibroblast-containing collagen type I hydrogels as follows: (1) keratinocytes only, (2) keratinocytes with melanocytes, (3) sweat gland cells. These substitutes were transplanted into full-thickness skin wounds on the back of immuno-incompetent rats and were analyzed after 3 and 8 weeks. Histological sections were examined with regard to myelinated and unmyelinated nerve fiber ingrowth using markers such as PGP9.5, NF-200, and NF-145. RESULTS: After 3 weeks, the skin substitutes of all three epidermal cell variants showed no neuronal ingrowth from the host into the transplant. After 8 weeks, we could detect an innervation of all three types of skin substitutes. However, the nerve fibers were restricted to the dermal compartment and we could not find any unmyelinated fibers in the epidermis. Furthermore, there was no distinct difference between the constructs resulting from the different cell types used to generate an epidermis. CONCLUSION: Our human tissue engineered dermo-epidermal skin substitutes demonstrate a host-derived innervation of the dermal compartment as early as 8 weeks after transplantation. Thus, our substitutes apparently have the capacity to attract nerve fibers from adjacent host tissues, which also grow into grafts and thereby potentially restore skin sensitivity

A SRY-HMG box frame shift mutation inherited from a mosaic father with a mild form of testicular dysgenesis syndrome in Turner syndrome patient

Background: Sex determining factor (SRY) located on the short arm of the Y chromosome, plays an important role in initiating male sex determination, resulting in development of testicular tissue. Presence of the SRY gene in females results in XY sex reversal and increased risk of gonadal germ cell tumours if the karyotype also includes the so-called GonadoBlastoma on the Y chromosome (GBY) region. The majority of mutations within the SRY gene are de novo affecting only a single individual in the family. The mutations within the high-mobility group (HMG) region have the potential to affect its DNA binding activity.Case Presentation: We performed G- and R-banding cytogenetic analysis of the patient and her family members including her father. We also performed molecular genetic analysis of SRY gene. Cytogenetic analysis in the patient (Turner Syndrome) revealed the mosaic karyotype as 45, X/46, XY (79%/21% respectively) while her father (milder features with testicular dysgenesis syndrome) has a normal male karyotype (46, XY). Using molecular approach, we screened the patient and her father for mutations in the SRY gene. Both patient and her father showed the same deletion of cytosine within HMG box resulting in frame shift mutation (L94fsX180), the father in a mosaic pattern. Histological examination of the gonads from the patient revealed the presence of gonadoblastoma formation, while the father presented with oligoasthenozoospermia and a testicular seminoma. The frameshift mutation at this codon is novel, and may result in a mutated SRY protein.Conclusion: Our results suggest that lack of a second sex chromosome in majority cells of the patient may have triggered the short stature and primary infertility, and the mutated SRY protein may be associated with the development of gonadoblastoma. It is of importance to note that mosaic patients without a SRY mutation also have a risk for malignant germ cell tumors

DNA Clasping by Mycobacterial HU: The C-Terminal Region of HupB Mediates Increased Specificity of DNA Binding

BACKGROUND: HU a small, basic, histone like protein is a major component of the bacterial nucleoid. E. coli has two subunits of HU coded by hupA and hupB genes whereas Mycobacterium tuberculosis (Mtb) has only one subunit of HU coded by ORF Rv2986c (hupB gene). One noticeable feature regarding Mtb HupB, based on sequence alignment of HU orthologs from different bacteria, was that HupB(Mtb) bears at its C-terminal end, a highly basic extension and this prompted an examination of its role in Mtb HupB function. METHODOLOGY/PRINCIPAL FINDINGS: With this objective two clones of Mtb HupB were generated; one expressing full length HupB protein (HupB(Mtb)) and another which expresses only the N terminal region (first 95 amino acid) of hupB (HupB(MtbN)). Gel retardation assays revealed that HupB(MtbN) is almost like E. coli HU (heat stable nucleoid protein) in terms of its DNA binding, with a binding constant (K(d)) for linear dsDNA greater than 1000 nM, a value comparable to that obtained for the HUalphaalpha and HUalphabeta forms. However CTR (C-terminal Region) of HupB(Mtb) imparts greater specificity in DNA binding. HupB(Mtb) protein binds more strongly to supercoiled plasmid DNA than to linear DNA, also this binding is very stable as it provides DNase I protection even up to 5 minutes. Similar results were obtained when the abilities of both proteins to mediate protection against DNA strand cleavage by hydroxyl radicals generated by the Fenton's reaction, were compared. It was also observed that both the proteins have DNA binding preference for A:T rich DNA which may occur at the regulatory regions of ORFs and the oriC region of Mtb. CONCLUSIONS/SIGNIFICANCE: These data thus point that HupB(Mtb) may participate in chromosome organization in-vivo, it may also play a passive, possibly an architectural role

Failure of SOX9 Regulation in 46XY Disorders of Sex Development with SRY, SOX9 and SF1 Mutations

In human embryogenesis, loss of SRY (sex determining region on Y), SOX9 (SRY-related HMG box 9) or SF1 (steroidogenic factor 1) function causes disorders of sex development (DSD). A defining event of vertebrate sex determination is male-specific upregulation and maintenance of SOX9 expression in gonadal pre-Sertoli cells, which is preceded by transient SRY expression in mammals. In mice, Sox9 regulation is under the transcriptional control of SRY, SF1 and SOX9 via a conserved testis-specific enhancer of Sox9 (TES). Regulation of SOX9 in human sex determination is however poorly understood.We show that a human embryonal carcinoma cell line (NT2/D1) can model events in presumptive Sertoli cells that initiate human sex determination. SRY associates with transcriptionally active chromatin in NT2/D1 cells and over-expression increases endogenous SOX9 expression. SRY and SF1 co-operate to activate the human SOX9 homologous TES (hTES), a process dependent on phosphorylated SF1. SOX9 also activates hTES, augmented by SF1, suggesting a mechanism for maintenance of SOX9 expression by auto-regulation. Analysis of mutant SRY, SF1 and SOX9 proteins encoded by thirteen separate 46,XY DSD gonadal dysgenesis individuals reveals a reduced ability to activate hTES.We demonstrate how three human sex-determining factors are likely to function during gonadal development around SOX9 as a hub gene, with different genetic causes of 46,XY DSD due a common failure to upregulate SOX9 transcription