Mercury is an extremely dangerous environmental contaminant responsible for episodes of human intoxication throughout the world. Methylmercury, the most toxic compound of this metal, mainly targets the central nervous system, accumulating preferentially in cells of glial origin and causing oxidative stress. Despite studies demonstrating the current exposure of human populations, the consequences of mercury intoxication and concomitant use of drugs targeting the central nervous system  are completely unknown. Morphine is a major option for pain management; its global consumption more than quadrupled in the last decade. Controversially, morphine has been proposed to function in oxidative stress independent of the activation of the opioid receptors. In this work, a therapeutic concentration of morphine partially protected the cellular viability of cells from a C6 glioma cell line exposed to methylmercury. Morphine treatment also reduced lipid peroxidation and totally prevented increases in nitrite levels in those cells. A mechanistic study revealed no alteration in sulfhydryl groups or direct scavenging at this opioid concentration. Interestingly, the opioid antagonist naloxone completely eliminated the protective effect of morphine against methylmercury intoxication, pointing to opioid receptors as the major contributor to this action. Taken together, the experiments in the current study provide the first demonstration that a therapeutic concentration of morphine is able to reduce methylmercury-induced oxidative damage and cell death by activating the opioid receptors. Thus, these receptors may be a promising pharmacological target for modulating the deleterious effects of mercury intoxication. Although additional studies are necessary, our results support the clinical safety of using this opioid in methylmercury-intoxicated patients, suggesting that normal analgesic doses could confer an additional degree of protection against the cytotoxicity of this xenobiotic. Mercury is a dangerous environmental contaminant. Methylmercury (MeHg), the most toxic compound of this metal, easily crosses biological membranes and tends to accumulate in tissues The central nervous system is the main target organ of this xenobiotic. The symptoms of acute human MeHg poisoning include ataxy, disartry, paresthesia, constraints of the visual field, mnemic deficits, and disturbances in the somatic sensory and hearing systems The molecular mechanisms elicited by MeHg exposure are not fully elucidated, but oxidative stress is closely related to toxicity and the neurodegeneration processes of mercury intoxication Oxidative stress also underlies other physiological responses, such as those elicited during drug treatments. However, the consequences of mercury intoxication and concomitant use of drugs targeting the central nervous system are completely unknown. This issue is especially important for drugs used in long-term treatments, such as analgesics. Morphine is one of the most potent analgesics, and possesses a recently demonstrated role in oxidative stress at therapeutic concentrations The analgesic effect of morphine is attributed to activation of a family of metabotropic receptors . Activation of the µ receptor, which is mainly responsible for morphine's effects, leads to the inhibition of calcium influx in pre-synaptic neurons and to an increase in potassium conductance in post-synaptic neurons, among other molecular effects. In addition to neurons, opioid receptors are localized in cells of glial origin, especially microglia and astrocytes, a feature that is preserved in gliomas like the C6 cell line A growing body of evidence suggests that morphine is active in oxidative stress, but despite morphine's extensive use, this role remains unclear and controversial 2 in Dulbecco's Modified Eagle Medium  supplemented with 10% (v/v) fetal bovine serum (CULTILAB), penicillin (50 U/mL), and streptomycin (50 µg/mL). Approximately 1.5×105 cells were seeded and maintained at 37°C for 24 h before exposure to MeHg, morphine, and/or naloxone.The rat glioma C6 cell line was obtained from the American Type Culture Collection  and maintained at 37°C and 5% COMeHg chloride (4 mM), morphine sulfate (10 mg/mL), and naloxone (4 mg/mL) were diluted in Dulbecco's Modified Eagle Medium. Cells were then incubated for 24 h with 0–11 µM, 0–10 µM, and 0–2.5 µM  of MeHg, morphine, and naloxone, respectively, in serum-free medium to avoid possible interference with serum proteins (MeHg shows high affinity for protein sulfhydryl groups).Cellular viability was quantified via the 4,5-dimethylthiazol-3,5-diphenyltetrazolium (MTT) method Levels of nitrites, a stable product of nitric oxide degradation, were analyzed with the Griess assay g. Absorbance of the supernatants was recorded at 535 nm and compared to measurements of standard concentrations of malondialdehyde.Levels of lipid peroxidation were analyzed with the thiobarbituric acid assay The levels of compounds containing sulfhydryl groups were assayed by the method described by Elman (1959) using the selective reaction of these groups with the acid 5,5′-dithium-bis(2-nitrobenzoic acid) (Elman's reagent) Total protein content was determined in all samples as described elsewhere 0 is the absorbance of the control group and A1 is the absorbance in the presence of morphine.To evaluate the possibility of direct scavenging of free radicals by morphine, the protocol described by Gülçin et al. http://www.mamiraua.org.br/pt-br/downloads/programas/). Analysis of variance was carried out, followed by a post hoc Tukey's test when appropriate. Differences with p<0.05 were considered significant.Statistical analysis was performed with BIOESTAT 5.3  in patients undergoing chronic treatment for pain management in clinical practice Here, the use of a high concentration of MeHg decreased cellular viability to ∼60%, but the therapeutic concentration of morphine reduced this deleterious consequence by ∼15% . These rMeHg intoxication was associated with a pronounced increase in nitrite levels . This obNitric oxide is a major free radical responsible for the oxidative damage resulting from MeHg exposure, leading to mitochondrial dysfunction and/or lipid peroxidation, among other effects What molecular mechanisms underlie this antioxidant action? To date, four possibilities were hypothesized for the antioxidant effect of morphine: 1) direct scavenger effect, 2) changes in levels of glutathione, 3) changes in activity of NADPH oxidase and 4) activation of opioid receptors see . InteresNo change in the levels of sulfhydryl groups was detected for any group . The higAlterations in glutathione levels following morphine treatment are frequently observed even at higher concentrations of the opioid Based on similar results obtained with a classical model of oxidative stress using hydrogen peroxide exposure In conclusion, this study demonstrated, for the first time, that a therapeutic concentration of morphine reduces the oxidative damage and cell death caused by MeHg intoxication through the activation of opioid receptors. Thus, these receptors may be a promising pharmacological target for decreasing oxidative damage and cell death after mercury intoxication. Although more studies are necessary, these results support the clinical safety of using morphine in MeHg-intoxicated people, suggesting that normal analgesic doses could confer an additional degree of protection against the cytotoxicity of this xenobiotic.Figure S1Cellular viability of C6 cell line exposed to increased concentrations of morphine for 24 h. No significant difference was detected between groups.(TIF)Click here for additional data file.


Conventional dentoalveolar osseous reconstruction often involves the use of graft materials with or without barrier membranes. To evaluate the efficacy of bone induction by bone matrix gelatin (BMG), delivered on an absorbable collagen sponge (ACS), compared to a placebo  in human alveolar socket defects. 20 alveolar sockets from 10 healthy adults were studied. In all cases, both the mandibular premolar area and the contralateral premolar area (as the control site) were involved. In each of the 10 patients, the extraction sites were filled randomly with BMG and ACS. The repair response was examined on day 90. Qualitative histological and quantitative histometric analysis, including the percentage of new-formed bone fill and density were done.Assessment of the alveolar bone indicated that patients treated with BMG had significantly (p<0.05) better bone quality and quantity compared to the controls. In addition, bone density and histology revealed no differences between the newly induced and native bone.de novo osseous formation for the bone regeneration.The data from this single-blind clinical trial demonstrated that the novel combination of BMG had a striking effect on  Dental rehabilitation of partially or totally edentulous ridges has become the primary indication for reconstruction or preservation of osseous structures to facilitate the insertion of endosseous implants. Unfavorable local conditions of the alveolar ridge following trauma, infection, developmental anomalies and surgical defects due to removal of pathologic lesions, may provide insufficient bone volume or unfavorable vertical, horizontal and sagittal intermaxillary relationships, thus rendering implant placement impossible or incorrect from a functional and esthetic viewpoint. Success rates are enhanced as increased bone volume accommodates utilizing longer and wider implants -3.Autogenous bone graft has been chosen among different methods for the reconstruction, as the ideal grafting material in bone reconstructive surgery, due to its osteogenic, osteoinductive and osteoconductive properties -6. AlthoRestoration and regeneration of a tissue depend on proliferation and the new matrix in the injured area. Recent studies have shown successful defect reconstruction by demineralized bone matrix or bone matrix gelatin (BMG), which contains bone constructing factors such as bone morphogenic protein (BMP) , 11. TheThe objective of this study was to evaluate the efficacy of bone induction by two BMGs delivered on an ACS compared to placebo  in human alveolar socket defect model following tooth extraction.Preparation of BMGet al (2006), formerly described a procedure for preparing BMG. We used a similar procedure with some modifications. Briefly, bovine bone was used as bone donors for BMG preparation. All soft tissues were removed and the bones were washed in sterile deionized water. The cleaned bones were kept for 1.5 h in a 1:1 mixture of chloroform and methanol (30 mL/g of bone), and then subjected to the following steps: 1) demineralization with 0.6 M hydrochloric acid (70 mg/g) for 18–20 h; 2) washing with sterile deionized water at pH 7.4; 3) washing with 2 M CaCl2 for 1 h at 21 °C; 4) washing with 0.5 M ethylene-ediamine-tetra-acetic acid for 1 h; 5) washing with 8 M LiCl for 1 h; and 6) washing with deionized water at 55 °C. The BMG was then incubated with Dulbecco’s modified eagle medium supplemented with 100 U/mL penicillin, and 100 mg/mL streptomycin for 1 h at 37 °C. The resulting BMG was lyophilized and stored in a desiccator at room temperature followed by sterilization with ethylene oxide, and degassing. To get a reasonable result with BMG graft, particle sizes of 74–500 µm should be resized into 200–500 µm. Therefore, after lyophilization, BMG pieces were put in a mortar and liquid nitrogen was added to it and pieces of bone matrix were chipped by pestle. The next stage was the use of standard 250–500-µm sieves to separate suitable sized particles. Then, an accurate scale was used to weight BMG in packages of 2 mg. BMG was packed in aluminum plates and was conserved at 70 °C until planted.Li, Patients and Surgical ProceduresThe experiment was done using 10 clinically healthy male and female patients. The patients aged between 20 and 60 years, and required contralateral tooth extraction in the mandibular premolar region and alveolar ridge preservation. They were all candidates for the present study and at the first visit, the patients were asked for their informed consent before enrollment in the study.The inclusion criteria were systemically healthy subjects who required extraction of a premolar tooth and residual extraction sockets with less than 50% bone loss in all dimensions. The exclusion criteria were the presence of severe periodontitis or acute infections at tooth extraction, pregnancy or planning to become pregnant within one year of the experiment, recent myocardial infarction or uncontrolled bleeding disorders, the presence of mental illnesses or suspected mental illnesses, hypersensitivity to bone graft materials, and the presence of clinically significant or unstable systemic diseases affecting bone or soft tissue growth, or other renal, hepatic, endocrine, hematologic, and autoimmune diseases.The present single-blind parallel-design randomized clinical trial was conducted at Tehran University of Medical Sciences, Tehran, Iran, from July 2011 to September 2013. The study protocol was approved by the Institutional Review Board, Tehran University of Medical Sciences.Surgical TreatmentAfter administration of local anesthesia, crestal and intrasulcular incisions were made to the adjacent teeth in all patients to expose the involved teeth and alveolar crest. Extractions were performed as atraumatically as possible. The teeth were sectioned if necessary to preserve all of the socket’s bony walls. The extraction sockets were thoroughly debrided to remove all of the soft tissue. The test material was delivered through a syringe and packed into the socket by one designated oral and maxillofacial surgeon. It was passively packed after careful bleeding control with gauze. The flaps were sutured over the materials using the mattress suture technique. Primary closure was obtained using periosteal releasing incisions, if possible; minor exposure was accepted.The medication prescribed to all subjects included antibiotics (500 mg amoxicillin 3 times daily for 3 days) and analgesics (200 mg ibuprofen 3 times daily for 5 days).eg, pain, discomfort, swelling, fever, and wound dehiscence)The oral wounds at the treated sites were examined at each visit, including at the baseline, days 2 and 14, and three months post-operatively, to monitor the occurrence of any of the commonly seen post-operative complications associated with the augmentation procedure . One mesial-distal central section (35-µm thick) from each defect was used for quantitative comparison of new bone formation. Histometric parameters were the percentage of bone fill and bone density in a field confined by the mesial, distal, and apical aspects of the surgically created defect and the coronal extent of induced or regenerated bone.Student’s t test for paired samples was used for the comparison of bone density and bone filling parameters between groups. Differences between each group during treatment time points were determined by Student’st test. A p value <0.05 was considered statistically significant.Statistics were presented as group means and SD. Clinical FindingsOnly one case at the site of ACS developed wound failure. Antibiotics and analgesics were used by subjects during the study. Other clinical complications such as hemorrhage, abnormal swelling, and infection were not seen in any patients during the follow-up visits.Bone FillThe percentage of bone fill showed differences in the rates and patterns of bone formation between the two treatment groups. The percentage of bone fill at specific time points showed significantly greater levels within BMG-treated defects than ACS-treated defects (p<0.05). Scattering of data in the group of BMG-treated defects was more than that in ACS-treated defects. The mean±SD percentage of bone fill of newly formed bone after 90 days in BMG-treated defects (27.83±3.07) was significantly (p=0.044) higher than that in ACS-treated defects (22.64±2.14).Bone DensityThe percentage of bone density after 90 days in BMG-treated defects (65.45±4.54) was significantly (p=0.004) higher than that in ACS-treated defects (59.43±4.00). Data distribution was almost similar in both groups but was slightly higher in the BMG-treated defects.Histological ObservationsIn the site of BMG, active remodeling of the surgical cavity was detectable in samples from the ACS sites, with normal lamellar bone structure, trabecular spaces and hematopoietic tissue. Active remodeling of the surgical cavity was detectable in samples from the BMG site, with normal cortical bone structure, bone trabeculae, and hematopoietic tissue. Newly formed bone showed recovery with normal characteristic. At the level of the medullary channel, no apparent alterations existed when the resident and healed bones were compared. From all aspects, the freshly formed bone in the BMG sites, exhibited qualities similar to that of the local bone or showed fairly dense trabeculation .Based on the studies that have shown a number of considerable advantages of BMG over other types of graft such as allogenous bone graft and other composite constructs, reconstruction of bone defects with an acid demineralized bone-derived material would be a potential treatment modality for bone formation .In the present study, we treated bone defects with an acid demineralized BMG and ACS to make a comparison between them. Histological analysis revealed rather similar or better bone healing response in BMG group. Regarding bone fill measurements, BMG showed consistently greater levels at every point in time (p<0.05).Other similar studies were performed on cats, as this study showed greater levels of bone fill at specific times within BMG-treated defects, which reached significance on days 14, 28, and 56. Regarding bone density, there was no significant difference between BMG and autogenous bone graft on days 14 and 28, but on day 56, bone density was significantly higher within the BMG-treated group. Similar to the present study, intra-treatment evaluation of bone formation in each group showed that bone density significantly increased during treatment, and bone fill in BMG group reached significance at every point in time. However, bone fill in the autogenous bone graft group only significantly increased from day 28 to 56 [Many authors have confirmed successful defect reconstruction using demineralized bone matrix or BMG, which contains many bone constructing factors such as BMP, but one study showed that producing human BMG to avoid bioincompatibility seems to be the next step for future studies to open a new horizon in the construction of bone defects.As a result of this study, BMG is an appropriate treatment option for the repair of bone defects that remain after surgery. The newly formed bone in the BMG implanted sites was not limited to the surface of the implanted gelatin; the new synthesized bone progressively extended in a vertical dimension along a net-like structure of BMG, which resulted in more formation of new bone. Moreover, BMG induced formation of bone trabeculae in close approximation to native bone so that it was difficult to distinguish between the implanted material and the newly formed bone.In conclusion, though limited by a small number of subjects, the results of this study demonstrated that the BMG had striking effect on new bone formation for the insertion of dental implants. The rate of bone reconstruction using BMG is higher than that for autograft and the quality is almost similar to normal tissue. In some cases, bone resulting from BMG has a better quality so that its density is more than the natural bone.


PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epeithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by  In vitro, various growth factors  mediated by cytokines and various growth factors tors EGF  have beeDiabetes mellitus is a chronic disease that leads to impaired healing resulting in chronic wounds, for which treatment and related complications are estimated to cost $10 billion annually. The pathophysiology of diabetic wound healing and development of new agents to improve clinical outcomes are continuously being investigated. In order to understand the mechanisms involved in impaired diabetic wound healing, and to test the efficacy and safety of new therapeutic agents, many animal models have been established and utilized. In particular, murine wound models have many advantages such as relatively low cost, ease of housing and handling that in turn support larger ‘n’ studies to increase statistical power. Excisional wound models in type 2 diabetic (db/db) mice have been widely utilized for investigations of impaired wound healing including evaluating the efficacy of topical PDGF-BB applied as a single agent or in combination with other cytoactive factors Skin contracture is the primary mechanism of wound closure in rodents; therefore the appropriateness of using mice for wound healing studies has been debated in vivo wound healing literature as well as the black-box warning for incidence of tumors with prolonged application of 0.01% PDGF-BB which is the currently used in clinics, an urgent need exists to determine the appropriate clinical indication for which a cytoactive factor is used in, and corresponding animal model to test the efficacy of that cytoactive factor in chronic wound healing and in diabetic wound management. To our knowledge, however, the effect of PDGF-BB on re-epithelialization or wound closure of splinted wounds in db/db mice has not been previously published. Motivated by the conflicting results in the literature regarding the efficacy of PDGF-BB in non-splinted murine wounds, the increasing popularity of splinted wound models in assessment of therapeutic effect, and the widespread clinical use of PDGF, the study reported herein was undertaken. In this manuscript, we report a controlled approach to monitor re-epithelialization and test the efficacy of PDGF-BB in accelerating wound healing in diabetic mice. The purpose of this study was to evaluate the effect of PDGF BB on wound healing by re-epithelialization and granulation tissue formation in db/db mice using a splinted wound model. We anticipate that these data will serve as critical reference for future studies investigating various drug delivery approaches for increased cytoactive factor retention.Considering the negative to modest effect of PDGF-BB in All procedures adhered to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for this study was approved by the Institutional Animal Care and Use Committee of the University of California, Davis (Protocol number: 17294). All surgery was performed under isoflurane anesthesia, and all efforts were made to minimize suffering.Spontaneously immortalized non-tumorigenic human keratinocyte cells (HaCaTs), generously provided by N.E. Fusenig and P. Boukamp and also catalogued with the American Type Culture Collection , were used for this study Human recombinant PDGF-BB  was dissolved in phosphate buffered saline  to obtain a stock solution of 100 µg/ml. This was serially diluted in serum free growth medium to obtain working concentration of 0, 7.5, 10, 50 ng/ml and 7.5 µg/ml. Cells (7000/well) were seeded in a 48 well plate in growth medium containing 1%(v/v) FBS and 1% (v/v) penicillin-streptomycin, and then treated with the various concentrations of PDGF-BB. Cell proliferation was measured after 5 days using the widely reported MTT assay Prior to seeding, cells were incubated with 10 µg/ml of goat polyclonal anti-PDGF receptor beta antibody for 30 min at 37°C on an orbital shaker. Cells were centrifuged at 1400 rpm for 5 min, the supernatant discarded and the pellet resuspended in growth medium and then plated in the presence of soluble of PDGF-BB as described above. Proliferation was determined after 5 days using the MTT assay.dbLepr; Jackson Laboratories, Bar Harbor, ME) were used for this study. The average ± SEM body weight of the mice was 52.6±0.4 g. Mice were housed four animals per cage pre-surgery and alone post-surgery; they were maintained in an animal care facility with a 12 hour light/dark cycle for four weeks prior to surgery and thereafter. Mice were randomly assigned to one of two treatment groups: 30 µl of 0.01% (w/v) PDGF-BB in vehicle  and 30 µl of vehicle control . The animal number was calculated with power and sample size analysis based on our previous study results. Alpha and beta levels were set at 0.05 and 0.8, respectively.Twenty-four genetically diabetic 16 week old male mice . Buprenorphine HCl  and lactated ringer’s solution  were administered subcutaneously following induction.The mouse was positioned in ventral recumbency on a temperature controlled heating platform . The dorsal surface was shaved with an electric clipper, sterilely prepped with 4% chlorhexidine and 70% alcohol gauze sponges, and then sterilely draped. All wounding procedures and post-operative treatments were performed by one surgeon. Prior to wounding, two sterile donut shaped splints  fabricated from 1.6 mm thick silicone sheet  were placed bilaterally around the pre-marked designated wound location , and then adhered with an immediate-bonding cyanoacrylate adhesive  and eight interrupted sutures using 6-0 nylon sutures . A full thickness wound was created inside each splint by using an 8 mm sterile skin biopsy punch and a Westcott scissor. A trimmed sterile plastic cover slip  was placed on top of the splint, and a semi-occlusive dressing  was applied circumferentially around the trunk of the animal. Animals were placed under a warming lamp and observed until they recovered fully from anesthesia.Body weight, appetite, symptoms for pain , and general health condition were monitored daily, and additional pain medication  was administered as needed. Mice were briefly anesthetized with isoflurane for about 5 minutes daily during bandage replacement, wound bed assessment, imaging, and treatment. The semi-occlusive dressing was cut open daily, along the coverslips placed on top of the splints, and the wound bed was evaluated for splint stability, inflammation, or infectious exudates. If excessive fluid accumulation was found, the fluid was gently wicked away with a cotton tip applicator. Wound beds were photographed on days 0, 2, 4, 6, 8, 10, and 11, with standardized exposure and focal length using a digital camera with macro lens and ring flash  mounted on our custom-made imaging station that provided incorporation of a ‘mm’ scale bar in each image obtained. Mice in the PDGF group received 30 µl of 0.01% PDGF-BB  dissolved in vehicle, filter-sterilized 5% PEG  in phosphate-buffered solution, while the vehicle control group received 30 µl of the PEG vehicle. Both wound beds in each animal were treated identically immediately after surgery and once daily for 10 days. Following evaluations and treatments, new sterile coverslips were placed over the splints and a piece of Tegaderm dressing  was applied circumferentially around the trunk of the mice. Bandages were replaced on days 2, 5, and 8. All mice were euthanatized with an intraperitoneal injection of Pentobarbital plus phenytoin  on day 11 following blood glucose measurement using a glucometer .:Photographs were analyzed to calculate percentage wound closure and percentage wound contraction using image analysis software  by a masked investigator. The outer and inner edges of wound closure Fig. 1 w% Wound Closure = (Wound Area on Day 0– Area of Inner Circumference on Day X)/Wound area on Day 0×100.Percentage wound contraction of each time point was calculated as:% Wound Contraction = (Wound Area on Day 0– Area of Outer Circumference on Day X)/Wound Area on Day 0×100.(see ).Note, the area of inner circumference excludes reepithilialization, while the area of outer circumference includes reepithelialization see Fig. 1All histopathological analysis was performed by a board certified veterinary pathologist who was unaware of experimental group for each sample. Following euthanasia, the entire wound and unwounded skin margins greater than 5 mm were excised to the depth of the retro-peritoneum. Samples were fixed in formalin for at least 24 hours and then sectioned through the center of the lesion to obtain the largest diameter of the wound. Tissue samples then underwent routine paraffin processing, were serially sectioned at a thickness of 5 µm and stained with hematoxylin and eosin, and picrosirius red. The widest section obtained was measured. The sections were photographed using a mounted digital camera  and images were analyzed using image analysis software  for length of reepithelialization, epithelial gap, amount of fibrovascular proliferation (granulation tissue) in the dermis, and inflammatory response.. Both sides of the lesion were measured and the final result was the sum of the two measurements. For wounds that had been completely re-epithelialized, a single measurement was taken from dermal edge to dermal edge. The epithelial gap was defined as the distance between the advancing edges of keratinocyte migration . Fibrovascular dermal proliferation (granulation tissue) was measured by examining the Picrosirius red-stained sections under polarized light which highlighted the newly deposited dermal collagen. Using the image analysis software the wound bed area was selected and the amount of collagen deposition in the selected area was automatically measured and was expressed as a percentage of the wound area . The inflammatory response was assessed using a semi-quantitative scoring system ranging from 0 to 4 where 0 indicates no inflammation, 1 indicates that 0–25% of the wound area was infiltrated by inflammatory cells, 2 indicates 25–50%, 3 indicates 50–75%, and 4>75% of the wound area was infiltrated.Length of reepithelialization was defined as the length of the layer of proliferating keratinocytes covering the wound area. This value was obtained by measuring the distance between the free edge of the keratinocyte layer and the base where the cells were still associated with native, non-affected dermal tissue . Both si. Both siIn our study, instead of subjectively estimating the area of apparent granulation tissue formation in the wound bed, we determined the collagen content in the whole wound by a semi-quantitative method. Preliminary studies verifying the validity of this approach demonstrated a correlation between a simple measurement of the area of peripheral granulation tissue with the quantitated collagen content. In our study collagen content in the wound bed was measured by quantifying the amount of Picrosirius red stained tissues under polarized light microscopy and using automated imaging analysis. We accurately determined the early fibroblastic reaction infiltrating the center of the wound in addition to the dense and well-formed granulation tissue at the edges of the wound bed. We believe that this method provides an accurate representation of the fibrovascular reaction associated with the healing process in the whole wound bed, especially in the diabetic mice where infiltration of fibrovascular tissue between the adipocytes is a feature Exclusion criteria included wound infection, spontaneous death, tissue destruction during processing for histopathological examination, and splint failure. Splint failure was defined as one or more of the following: splint fracture; partial or complete detachment of splint; two or more sutures missing or released. When one missed or released suture was found during daily examination, the suture was restored.in vitro proliferation assay, statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. The effects of mouse strain, presence of the splint, dressing, treatment, time, and interaction between time and each effect on the body weight, wound healing, and contraction were evaluated using repeated measures ANOVA. Histological scores were compared between the groups by using the Student’s t-test. Values of P<0.05 were considered significant.Results are expressed as mean ± SEM. Data from individual mouse bilateral wounds were averaged and considered as one data point. If one of the two wounds were excluded during the experiment based on exclusionary criteria, data from only the inclusionary wound was used as the data point for comparisons. Statistical analysis was performed using a commercially available statistical software SPSS . For the . Proliferation in treated cells, while remaining higher than in untreated cells, appeared to decrease between 7.5 ng/ml to 15 ng/ml before again increasing. When the cells were blocked with an antibody to the PDGF beta receptor, proliferation was significantly reduced for all concentrations, approximating results obtained for untreated cells . HaCaT cells treated with PDGF-BB did not show any demonstrable significance in proliferation rates (data not shown).Proliferation was significantly higher in PDGF-BB treated fibroblasts  compared to untreated cells. The dose response was trimodal in nature. Maximum proliferation was observed for the following doses: 7.5 ng/ml, 50 ng/ml and 7.5 µg/ml . Prolife. Mean ± SEM percentage wound contraction was 22.6±1.9% in the PDGF group, and 22.0±4.7% in the vehicle group . In overall comparisons, no significant effect of treatment on wound contraction or closure between the PDGF and VEHICLE groups was observed . In histopathological evaluations, there were no significant differences in epithelial gap (P = 0.74) or reepithelialization (P = 0.47) . There were also no significant differences in collagen content (P = 0.62) or inflammation score  were excluded from the study based on our strict exclusion criteria. However we were able to use data obtained from the contra-lateral wounds, therefore all animals (24/24) were included for data analysis. Mean ± SEM percentage wound closure on day 11 was 86.1±6.2% in the PDGF group and 89.7±5.3% in the vehicle group . Mean ± . Mean ± . Mean ± . Mean ± In this study, the applicability of a splinted wound model to ascertain the ability of PDGF-BB in facilitating wound closure was evaluated. The results of this study would become baseline data for using the splinted wound model to evaluate advanced technologies developed to improve delivery of PDGF-BB for the treatment of chronic wounds.in vitro cell proliferation assays. In vitro, PDGF-BB significantly accelerated cell proliferation in a trimodal pattern with maximum effects observed at 7.5 µg, 50 ng and 7.5 ng in this study . Blocking PDGF receptor beta significantly inhibited the proliferative ability PDGF-BB. In vivo, significantly accelerated wound closure was demonstrated in previous studies in vitro studies and, a dose of 3 µg per wound (8 mm diameter) was chosen for this study, which corresponds to approximately 6 µg/cm2. Wounds were treated daily for 10 days after wounding. Considering that the wounds in our study were smaller (at least one fourth the wound area compared to other studies) we chose to monitor wound closure over 11 days after wounding instead of the 21 days for the larger wounds previously reported The bioactivity and specificity of PDGF-BB in facilitating cell proliferation was determined using 2 wound area) for 5 and 10 days. However, they did not observe any differences in wound closure up to 10 days after wounding for treated groups. Other studies Previously published reports have shown variability in efficacy of PDGF-BB in promoting closure of nonsplinted wounds in db/db mice depending on experimental conditions such as PDGF dosage, treatment duration, and study endpoint in vivo by chemotactic and mitogenic promotion of granulation tissue formation and reepithelialization Another difference between previous studies Another factor that can impact wound closure is the dressing methods. The importance of application and maintenance of semi-occlusive dressings, and infection control have been discussed previously in vitro using fibroblasts and keratinocytes. Our in vitro results demonstrate that PDGF-BB did not accelerate proliferation of HaCaTs, but effectively increased proliferation of primary human foreskin fibroblasts. The failure to promote proliferation of HaCaTs in response to PDGF-BB was anticipated as they inherently lack PDGF beta receptors In vivo the baseline expression of PDGF receptors have been reported to be significantly inhibited in genetically diabetic db/db mice; expression of the B-type receptor is further inhibited during repair in vivo expression of PDGF receptors in our study, we can relate our findings and infer from previous studies that the expression of PDGF B receptors, required for a PDGF-BB specific response, is significantly inhibited. This may lead to inhibition of both the autocrine and paracrine signaling effects of PDGF required for healing. We also recognize that the expression and activities of matrix metalloproteinases and other proteases are altered during diabetes and may contribute to accelerated degradation of growth factors db/db mice) upon PDGF treatment using the splinted model.The bioactivity of PDGF-BB used in the study as confirmed Advanced protein glycosylation is a process of non-enzymatic modification of proteins and plays a significant role in ageing and diabetic pathology. Advanced glycation end products (AGE) generated as a result of this process can exacerbate the disease process via oxidative damage, apoptosis and inflammation, and is implicated in Alzheimer’s disease, cardiovascular disease and diabetes mellitus to name a few in vivo needs to be carefully interpreted with critical consideration of the wound model used and there is much remaining to be understood about the effect of growth factors for treatment of chronic wounds, especially in disease models.The results of this study demonstrated no detectable effect of PDGF-BB on wound healing in db/db mice with splinted full thickness wounds. A species specific difference in the usage of human recombinant PDGF-BB to treat chronic wounds in mice is highly unlikely considering that PDGF-BB is mitogenic in murine cells


We read with great interest the article of Bayani et al. on the “Vitamin-D status in diabetic patients” published in Caspian Journal of Internal Medicine (CJIM) . Bayani The study reported the prevalence of risk factor for both the lowest quartile <21 ng/mL) and the highest quartile ≥31 ng/mL) of serum vit D levels in the study population. Women, Iranian people and older people were most likely to have low vit D levels. Several studies showed that two previous decades displayed a high prevalence of vit D deficiency in tropical countries such as China, Turkey, India, Iran and Saudi Arabia and estimated its prevalence between 30% and 93% (1 ng/mL a1 ng/mL oMelanin in the darker skin reduces the ability to make vit D in response to sunlight exposure, because it absorbs the sunlight , 5. The


Rift Valley fever virus (RVFV) remains heavily neglected in humans in Mozambique, even though recent outbreaks were reported in neighboring countries in humans and several cases of RVFV in cattle were reported in several districts in Mozambique.We conducted a cross sectional study during and after severe flooding that occurred in 2013 in Mozambique. Paired acute and convalescent serum samples were tested from febrile patients attending a primary health care unit in a suburban area of Maputo city for the presence of IgG and IgM antibodies against Rift Valley fever virus (RVFV) using enzyme-linked immunosorbent assay (ELISA). Seroconversion of IgG anti-RVFV was observed in 5 % (10/200) of convalescent patients and specific IgM anti-RVFV was detected in one acute patient . All sera from acute patient tested negative by real time PCR.In conclusion, our results suggest that RVF represent an important but neglected cause of febrile illness following periods of flooding in southern Mozambique. Rift Valley Fever Virus (RVFV) is a mosquito borne zoonotic virus that has emerged as an important cause of febrile illness in several sub-Saharan countries –5, incluRecent publications demonstrated that anti-RVFV IgG was often detected in cattle in Maputo and Gaza Province, both situated in the southern Mozambique –11, and 2 and a total population of 293 361 habitants  and age > 5 years were invited to participate. Written consent was requested prior to enrollment. This study was approved by the National Bioethical Committee in Mozambique (Ref: IRB00002657) prior to initiation. Exclusion criteria included, pregnancy, presence of psychiatric disease and febrile disease with a readily identifiable focus of infection, such as otitis media, sinusitis, purulent pharyngitis, cellulitis, urinary tract infection, dental abscess, septic arthritis, pneumonia or pelvic inflammatory disease.3EDTA tube (5 ml) and into serum separation tube . All participants were requested to return to the health facility three weeks later for follow up and collection of additional 5 mL of blood  into K3EDTA tube.A total volume of 10 mL of blood was requested from each volunteer during the enrollment visit (acute sample) and collected into KA standardized questionnaire was used to collect clinical, demographic and epidemiological data, including information on risk factors for RVFV infection.Plasmodium falciparum, P.malarie, P. ovale and P. vivax using light microscopy. Parasite density was estimated by means of a semi-quantitative scale [Acute sample drawn from each participant was tested for malaria using blood smear microscopy. A thick blood smear was mounted from anticoagulated whole blood. All blood smears were stained using the Giemsa protocol and screened for ve scale .Samples were shipped and tested at The Center for Emerging and Zoonotic Diseases of the National Institute for Communicable Diseases, National Health Laboratory Service (NICD/NHLS) in South Africa. The testing algorithm was as follows, at first, all convalescent serum samples were initially tested using recombinant nucleoprotein (rNP)-based anti-RVFV IgG ELISA . If the For ELISA testing, we followed strictly the instructions described in published literature and details of the testing procedures and interpretation of IgM and IgG assays, are described in the two published manuscripts , 19. Thep value < 0.05 was considered of statistical significance.Data analysis was performed using the statistics package STATA 9.0 . Simple frequencies were calculated for each study variable. Study groups were compared using Kruskal Wallis test. Associations between categorical variables were determined using logistic regression analysis. A Three hundred and seventy five patients were enrolled between January and September 2013 and 175 did not return to their convalescent visit  and 56.7 % (98/173) were female.Of the 200 convalescent samples, 10 % (20/200) tested positive for IgG anti-RVFV. Seroconversion for IgG anti-RVFV was confirmed in 10  samples. Most of samples from patients with serological evidence of acute infections , were clustered between February and April with a peak in April  acute RVFV infection if they seroconverted for anti-RVFV IgG antibodies between the acute and convalescent visit 10/200, 5.0 %), ii) previous RVFV exposure if they presented anti-RVFV in both the acute and convalescent visit  and iii) no anti-RVFV antibodies . Table 00, 5.0 %Aedes mosquitoes were abundant [To our knowledge this is the first investigation of RVFV among febrile patients in Mozambique and we found that 5 % 10/200) of febrile patients seroconverted to IgG antibodies against RVFV between their acute and convalescent samples. Although novel for Mozambique, this finding is not surprising, based on the following arguments: i) A recent study conducted by our group in the same area showed that abundant  ii) rece/200 of fabundant , iii) seabundant , 8, 23, abundant , v) peopabundant  and vi) abundant . AlthougOnly 1 patient was positive for anti-RVFV IgM antibodies in the acute sample. This may be explained by the fact that the average number of days between onset of fever and blood collection was one day, and two patients were enrolled two days post onset of fever, likely, before the seroconversion of IgM antibodies. IgM anti-RVFV was not performed in the convalescent samples and we acknowledge this as a limitation of our study, but the demonstrated seroconversion for IgG antibodies against RVFV should be regarded as evidence for recent exposure to RVFV. The anti-RVFV IgG ELISA was intensively validated according to the results of a virus neutralization test in 2967 sera collected in Africa and was shown to have diagnostic sensitivity and sensitivity close to 100 % , 19.PCR results were all negative. Negative PCR results might be attributed to the fact that viremia in these patients is transient. We also argue that PCR results might be explained by the fact that serum samples were poorly handled, by repetitive and multiple freeze-thaw cycles and long periods of storage at room temperature, resulting in degradation of the RNA or viral particles.No cases of severe diseases was reported and this might be explained by the fact that patients were recruited at the outpatient triage at the primary health care facility, where the majority of patients presents with non-severe disease.Of remark, our data showed that 67 % of patients with serological evidence of acute RVFV infection and negative malaria blood smear were treated with antimalarial drugs. This demonstrates that most of RVFV infections are misdiagnosed as malaria.In conclusion, our data suggests that RVFV infection occurs among febrile patients in the periods of heavy rainfalls and flooding in southern Mozambique and that most of cases are misdiagnosed as malaria, leading to mistreatment with antimalarial drugs.


Data is sparse concerning the magnitude of metabolic syndrome (MetS) in developing countries like Ethiopia whose major health problem had long been under-nutrition and infectious diseases rather than non-communicable diseases (NCDs) including hypertension, obesity and MetS. However, it is obvious that the NCDs are recently taking over and becoming the major health care concerns in the developing countries. This pattern could be partly explained by the nation’s sustained economical growth in the last few decades in addition to the increasing globalization related adoption of western lifestyle. The aim of this study was to assess the prevalence of metabolic syndrome and associated factors among hypertensive patients in North West Ethiopia.A cross sectional study was conducted on 300 hypertensive individuals who get follow-up care at University of Gondar Hospital after diagnosed as hypertensive. The WHO STEP-wise approach to surveillance of NCD was used. Fasting blood glucose level, triglyceride and high density lipoprotein cholesterol were determined using standardized laboratory procedures.The prevalence of metabolic syndrome was 40.7% and 39.3% according to the modified NCEP-ATP III and IDF criteria respectively. Low HDL-c was found to be the most frequently encountered (81.3%) component of MetS other than the hypertension. Being female was significantly associated with MetS  using IDF and  using NCEP-ATP III criteria. Abnormal BMI which included both high and low BMI was found to have significant association with MetS  using IDF and  as diagnosed using the NCEP-ATP III criteria.We recommend a comprehensive medical care approach to hypertensive patients to adequately assess and address the additional components of MetS which are known to potentiate the risks of cardiovascular diseases among hypertensive patients. Hypertension and Metabolic syndrome (MetS) are highly prevalent diseases that present a global challenge , 2. In 2When hypertension and MetS components co-exist in an individual they potentiate one another leading to synergism that increase the total CVD risk . HyperteEven though there are different studies done on the prevalence of metabolic syndrome in different parts of the world –10, dataThis study was conducted in the University of Gondar hospital, from July 1 to August 31, 2013. The hospital has more than 500 beds capacity and serves as a tertiary level referral center for over five million people in and around Gondar town. This cross sectional study was conducted among hypertensive patients having follow-up care at the hypertension clinic of the hospital which is scheduled as a day weekly service at chronic illness clinic.The sample size was estimated based on single population proportion formula using a confidence interval (CI) of 95% and a 31.2% previous prevalence of metabolic syndrome among hypertensive patients from Nigeria .Where, n = sample size, p = proportion of hypertensive patients who may have metabolic syndrome and d = assumed marginal error. Based on the above formula and assumptions, the sample size was calculated to be 300. Then, every third participants were selected using systematic random sampling technique. There were a total of 3000 registered hypertension patients attending the clinic with an average follow-up interval of 2-3 month. All participants were with the age of greater than or equal to 18 years.The socio-demographic, behavioral, physical and clinical characteristics of the study participants were collected using an interview based structured questionnaire adopted from the WHO STEPS manual that was prepared for this purpose . Data thAnthropometric data  were collected according to WHO STEPS manual . Height Blood pressure (BP) was measured using a standard adult arm cuff of mercury type sphygmomanometer after 10 min rest in the clinic by the nurses working in the chronic diseases follow up clinic. To improve the reliability of measurement two readings were taken with 1 min interval and the average of the two readings was recorded as the final BP of the patient. But if the difference between the two readings was greater than 5 mmHg, a third measurement was taken and the average of the three readings was recorded as the final BP of the patient .Five milliliter of venous blood was collected from each patient in plane test tubes and serum was separated immediately. The extracted serum was investigated for High density lipoprotein-cholesterol (HDL-c), triglyceride and fasting glucose using Mindray BS-200 chemistry analyzer  according to manufacturer’s instructions.Triglycerides was determined by enzymatic hydrolysis with lipase , HDL-c was determined by specific enzymatic method after eliminating chylomicrons, very low density lipoproteins and low density lipoproteins  and glucose was determined by glucose oxidase method .Metabolic syndrome was defined according to modified NCEP-ATP III and IDF criteria.≥94 cm for men and ≥80 cm women) plus two of any of the following risk factors: (1) raised TG level (≥150 mg/dL); (2) reduced HDLC ; (3) raised blood pressure (systolic BP ≥130 or diastolic BP ≥85 mmHg) or treatment of previously diagnosed hypertension; (4) raised FG (≥100 mg/dL) [In accordance with the IDF criteria, subjects were classified as having MetS if participants had abdominal obesity (defined as waist circumference of 0 mg/dL) .>102 cm in males and >88 cm in females); (2) hypertriglyceridemia (TG ≥150 mg/dL); (3) reduced HDL-c ; (4) high BP (≥130/85 mmHg); (5) FG (≥100 mg/dL) [In accordance with the ATP III criteria, subjects were classified as having MetS if participants had three or more of the following risk factors: (1) abdominal obesity (waist circumference 0 mg/dL) .± standard deviation or median (inter quartile range) and categorical variables were expressed as number (percentage). Univariate and multivariate binary logistic regression analysis was used to evaluate the differences in the distribution of categorical variables for study groups. P-value < 0.2 was used as a cutoff to include variables for multivariate binary logistic regression model. Student’s t-tests was used to evaluate differences in mean for study groups. The prevalence estimates for metabolic syndrome according to the two definitions was determined separately. In all cases P-values < 0.05 at 95% confidence level was considered statistically significant.Data was entered into EPI INFO computer software and exported to SPSS version 16 for analysis. Frequency distributions of socio-demographical, clinical and behavioral characteristics of subjects were explored. Continuous variables were expressed as mean Trained nurses working in the chronic diseases follow up clinic were involved in the collection of socio-demographic, behavioral, clinical and physical characteristics from the patients. The proper functioning of instruments, laboratory reagents, and technical performance was checked by using quality control samples. Standard operating procedures (SOPs) were followed starting from sample collection up to result reporting. All laboratory procedures were handled by laboratory technologists.Ethical clearance was obtained from School of Biomedical and Laboratory Sciences, University of Gondar. Before enrolling any of the eligible study participants; the purpose, benefits and risk of the study was clarified and discussed for each participant. Data was collected after informed consent was obtained and confidentiality of the information was kept by using codes. The results of the participants were communicated with the clinicians for better management of the patients.A total of 300  hypertensive patients, with a mean (±SD) age of 54 (±14) years, participated in this study. Majority, 78.3%, 56%, 41% of the study participants were urban dwellers, married and educationally unable to read and write respectively. Only 8.3% of the study participants had alcoholic drink at least once in the previous year and only 1% of the participants had a history of smoking. Palm oil was used by 55% of the study participants to prepare food. The 21.7% of the study participants had a day to day working habit full of vigorous intensity of activities which includes farming; long distance walking, laborious activities. Only 6% of the study participants have history of performing a purposely planned regular physical exercise  and 94.7% started antihypertensive drug therapy. Among the patients who started treatment 42% were taking at least two types of antihypertensive drugs. The median (IQR) time since the initiation of treatment was 2 (0.4-4) years for men and 2 (0.5-6) years for women. Abnormal body mass index, which in this study includes both high and low BMI, was observed among 40% (121/300) of the study participants; women (45.4%) being more frequently affected as compared to their male (32.2%) counterparts. Women also showed higher mean concentrations of glucose (93.3 mg/dl) and HDL cholesterol (37.8 mg/dl). The median triglyceride values were 107 mg/dl for men and 115 mg/dl for women  hypertensive patients had abdominal obesity among whom 118 (94.4%) fulfilled two additional metabolic syndrome components. Hence, the overall prevalence of MetS according to the IDF criteria was 39.3%; 24.3% among men and 48.6% among women. The frequency of metabolic syndrome components, other than the hypertension, according to the modified NCEP-ATP III criteria was 18.7%, 25.3%, 27.3% and 81.3% for abdominal obesity, elevated fasting glucose, elevated triglyceride and reduced HDL-c respectively. Higher prevalence of abdominal obesity and reduced HDL-c were observed among women than men. These values were similar applying IDF criteria except for abdominal obesity which the prevalence was 58.3% with significantly higher values among women (51.9%) compared to men (25.2%) , being a government employees , being unable to read and write , having a monthly income of 601-1500 Ethiopian birr , having abnormal BMI  and using palm oil for food preparation  were found to be significantly associated with the development of MetS. However, on the multivariate analysis the association was maintained for being female , being unable to read and write  and abnormal BMI . Additionally, some variables that didn’t show significant association on the univariate analysis such as duration since hypertension diagnosis 1-5 years , duration since hypertension diagnosis >5 years , duration since on anti-hypertensive therapy 1-5 years  and duration of anti-hypertensive therapy >5 years  were found to have significant association , being a government employees , having abnormal BMI , having an educational status of less than primary school , living in the rural area , duration since hypertension diagnosis > 5 years  and duration since ant-hypertensive therapy > 5 years  were found to be significantly associated with the development of MetS. However, the multivariate analysis showed that being female , having an educational status of primary school , being unable to read and write  and abnormal BMI  have maintained the significant association with MetS . Moreover, females were found to have a higher prevalence of metabolic syndrome  as compared to men . Though not statistically significant, increased occurrence of metabolic syndrome was also observed in the subjects aged ≥50 years.A study from Nigeria has reported a metabolic syndrome prevalence of 31.2% among newly diagnosed hypertensive patients according to the NCEP-ATP III criteria and a study from Kuwait reported a prevalence of 34% , 9. ThesThe frequency of metabolic syndrome components in this study was 18.7%, 25.3%, 27.3% and 81.3% for abdominal obesity, elevated fasting glucose, elevated triglyceride and reduced HDL-c respectively. All these values, except for reduced HDL-c, were lower than a study conducted in Jordan . This miConcordant to our finding, the association between sex and metabolic syndrome was reported in other studies , 21. IndOur study has indicated that having an educational status of less than of less than primary school level has significant association with the occurrence of Mets. This could be explained by the fact that the lifestyle, type of work and even place of residence could be affected by the level of education which may also indirectly impact the risk and occurrence of MetS. This could also partly explain the effect of income on the occurrence of MetS.Studies from Nigeria  and KuwaDue to the cross sectional nature of the study, temporal relations could not be established between metabolic syndrome and the associated factors. Moreover, only two definitions were used to assess the prevalence of MetS; a different prevalence rate could have been observed if other metabolic syndrome definition like the WHO definition were used. Despite these limitations this study ultimately adds to the limited data on metabolic syndrome in sub-Saharan Africa, particularly in the study area.The prevalence of metabolic syndrome among hypertensive patients is high, with low HDL-c being the most commonly encountered abnormality. Being female, having abnormally high or low BMI and having less than primary school educational status were found to have significant association with the occurrence of MetS. Majority of the hypertensive individuals were at a high risk of developing MetS as they have one more very common additional risk factor (low HDL-c). Therefore, all hypertensive patients should be aggressively screened for the presence of additional cardiovascular risk factors that constitute the metabolic syndrome. Management of hypertensive patients in the presence of an additional component of metabolic syndrome should be comprehensive enough to address the increased risk of cardiovascular and diabetes related events. Moreover, further in depth studies should be conducted to assess predictors of MetS and the performance of the health care system in order to design, and implement a cost-effective and comprehensive patient care approach for hypertensive patients.


Patient was found to have mucous membrane pemphigoid involving the oropharynx and extending to trachea, till just above main stem carina. Four other cases described mucosal pemphigoid involving the trachea. We hereby present a brief review of current consensus on management of mucous membrane pemphigoid with airway involvement. Mucous membrane pemphigoid is a chronic inflammatory autoimmune disease process that is characterized by subepithelial blistering, primarily of the mucosa, with linear deposition of Immunoglobulin G (IgG), Immunoglobulin A (IgA), and Complement 3 (C3) at the basement membrane . UnfortuWhile there have been a number of case reports of mucous membrane pemphigoid, less than a handful of the cases involve the trachea –7. This 2) of 44.3 mm Hg, and partial pressure of oxygen (PaO2) of 57.8 mmHg. She was also found to have a white blood cell count of 28.8·109/liter and a hemoglobin level of 12.2 g/dL. Chest X-ray demonstrated cardiomegaly with prominence of pulmonary vessels. Computed tomography of the chest demonstrated infiltrates in bilateral lower lobes, right greater than left, consistent with pneumonia ( Staphylococcus aureus (MRSA), which responded to the treatment with vancomycin.A 34-year-old African American female patient with a history of obstructive sleep apnea, Graves disease, and mucocutaneous pemphigoid with corneal and oropharynx involvement presented to our emergency department (ED). She was diagnosed by the biopsy of her corneal lesion and immunofluorescence studies which was consistent with the diagnosis of mucous membrane pemphigoid. In past she was treated with methotrexate and prednisone. She developed pneumonia while being on methotrexate, which was then stopped. Later she was given rituximab due to her progression of disease for 3 months. She presented to our emergency department (ED) with a 1-week history of malaise, lethargy, nausea, and productive cough with thick yellow sputum and approximately 25 mL of hemoptysis daily. In the emergency ED, the patient was found to have conversational dyspnea with severe wheezing. She was placed on oxygen via nasal cannula at 5 L/min and anticholinergic and antimuscarinic medication via nebulizer therapy for bronchodilatation, followed by intravenous methylprednisolone, vancomycin, and piperacillin/tazobactam. Arterial blood gas showed a pH of 7.45, partial pressure of carbon dioxide  , 10. AddThere are few cases of tracheal involvement in patients with pemphigoid. Hemoptysis or respiratory compromise in a patient with mucous membrane pemphigoid should prompt the clinician to investigate whether the patient has tracheal involvement and consider intensification of therapy. More research is needed to compare targeted humoral immunosuppressive therapy with corticosteroids or disease modifying antirheumatic drugs (DMARDs) such as cyclophosphamide or azathioprine for both induction and maintenance of remission.


Roseobacter, Vibrio, Bacteriovorax, and Lacinutrix that were distant relatives of genomes already described from marine habitats. Besides, using assembly from deep metagenomic sequencing from the particle-specific enrichments we were able to determine a total of 20 groups of contigs (eight of them with >50% completeness) and reconstruct de novo five new genomes of novel species within marine clades (>79% completeness and <1.8% contamination). We also describe for the first time the genome of a marine Rhizobiales phage that seems to infect a broad range of Alphaproteobacteria and live in habitats as diverse as soil, marine sediment and water column. The metagenomic recruitment of the communities found by direct sequencing of the large size filter and by enrichment had nearly no overlap. These results indicate that these reconstructed genomes are part of the rare biosphere which exists at nominal levels under natural conditions.We have used two metagenomic approaches, direct sequencing of natural samples and sequencing after enrichment, to characterize communities of prokaryotes associated to particles. In the first approximation, different size filters (0.22 and 5 μm) were used to identify prokaryotic microbes of free-living and particle-attached bacterial communities in the Mediterranean water column. A subtractive metagenomic approach was used to characterize the dominant microbial groups in the large size fraction that were not present in the free-living one. They belonged mainly to Actinobacteria, Planctomycetes, Flavobacteria and Proteobacteria. In addition, marine microbial communities enriched by incubation with different kinds of particulate material have been studied by metagenomic assembly. Different particle kinds  were colonized by very different communities of bacteria belonging to  Characterizing the true diversity of microbes in nature is a daunting task, yet great advances have been achieved through the use of high throughput sequencing  and large (5.0–20.0 nμm) filter fractions theoretically representing the FL and PA communities, respectively. We also investigated the selective impact of different particles on community composition using metagenomic sequencing of particle-specific enrichments. Our results confirm that the Mediterranean FL and PA microbial communities are distinct. Moreover, we show that different particle chemical composition and structure can drive the enrichment of distinct and specific particle-associated community structures. In addition, using assembly from deep metagenomic sequencing from the particle-specific enrichments, we describe several nearly complete genomes most of them belonging to new species. These reconstructed genomes are part of the rare biosphere, which exists at nominal levels under natural conditions.For the free-living  and particle-associated  metagenomes, 200 L of Mediterranean seawater was collected off the coast of Alicante, Spain (38°4′6.64″N/0°13′55.18″W) on September 6th 2013 and January 27th 2015 from 55 and 20 m depth, respectively (16°C). The seawater was sequentially filtered using 20, 5, and 0.22 μm filters , and the filters were immediately frozen at −20°C until processed for DNA extraction. For the particle-enriched (PE) metagenomes, seawater was also collected off the coast of Alicante, Spain (38°16′42″N/00°14′39″W) on May 20th 2013.In the laboratory, 480 mL of seawater from 55 m depth was added to each of five flasks (2L) containing 50 g of one of the following: 1. seawater from the above sampling—control (SW), 2. Sand , 3. Diatomaceous Earth , 4. Cellulose , 5. Chitin  autoclaved and dissolved in 20 mL. Low levels of pyruvate  and yeast extract  were also added to each sample, which were then incubated for 2 weeks at 15°C with 12 h light/dark cycles with constant stirring. In order to collect particle-associated microorganism after these 2 weeks of incubation each particle-enriched culture (500 ml) was filtered through a 20 μm filter, allowing sea water to flow through and retaining only the particles and any bacteria attached to them. This was followed by 3 washes, in which 300 mL of sterile sea water was added to the particles. It should be noted that the control sample (SW), which did not contain any particles, was not subjected to any treatment until the end of the 5 weeks incubation. The whole sample was spun at 13,500 rpm for 30 min to pellet the microbial cells. A schematic representation of the enrichments experimental design is shown in Figure de novo assembler . Gene predictions on the assembled contigs were performed using Prodigal , using the 0.22 and 5 μm filters, respectively. For the PE metagenomes, community DNA extractions were performed using either pelleted cells (SW) or rinsed and filtered particles . In all cases, 5 mL of lysis buffer  were added to the biomass  and incubated at 37°C for 45 min with 50 μL Lysozyme [100 mg/mL]. Nucleic acids were extracted with phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol and DNA integrity was checked by agarose gel electrophoresis. The metagenomes were sequenced using the Illumina Hi-Seq 2000 (100-bp paired-end read) sequencing platform by GATC Biotech . The IDBA-UD Phylogenetic classification of the metagenomic data was performed using three different methods. In the first, ribosomal RNA (rRNA) sequences were used as queries for BLASTN searches against the entire RDP database and classified into a high level taxon if the sequence identity was ≥80% and the alignment length was ≥90 bp. For the second method MetaPhlAn version (v2.0) was used to study the microbial community  that provide a normalized value comparable across various metagenomes  and MedDCM-SEP2013-LF (BioSample SAMN02954152), respectively. FL-Winter and PA-Winter metagenomes have been submitted as MedWinter-JAN2015-20m (BioSample SAMN04874669) and MedWinter-JAN2015-20m-LF (BioSample SAMN04880534), respectively.a] concentration (mg m−3) was only slightly higher during the summer sampling (0.92) compared to the winter sampling (0.75) but similar to the average [Chl a] observed at the DCM depth in different points in the southwestern Mediterranean Sea that reaches 0.88 mg m−3 . Interestingly, however, the summer PA fraction had a bimodal GC distribution  independent of the season. These results are similar with previous observations in particles enriched metagenomes  metagenomes sequenced from either the 0.22–5.0 μm (FL) or the 5.0–20.0 μm (PA) filter fractions of two separate samples. One sample was taken during the summer from the deep chlorophyll maximum (DCM), a zone of maximal phytoplankton concentration that generally occurs below the thermocline. In tropical waters this is a permanent feature, whereas in the Mediterranean and other temperate waters the DCM is a seasonal phenomenon (summer and part of spring and autumn). The second sample was collected during the winter mixing that occurs in January. However, the  and TARA 93 from South Atlantic Ocean [5 and 30 m]) from the FL fraction in the TARA metagenomes. The lack of recruitment observed here suggest that particle enrichment, like many other culturing techniques, selects for a small subset of microorganisms that flourish in these specific conditions but do not fare so well in more complex natural environments. These kinds of microbes which exist at nominal levels under natural conditions are part of the denominated “rare biosphere” (Sogin et al., To examine the distribution of the novel enriched microorganisms described above we performed fragment recruitment analysis by comparing each contig group to that of numerous metagenomes from a variety of seawater samples (DeLong et al., Ca. Pelagibacter, are collected in the large pore size, likely as a result of clogging that produces a much smaller pore size. In addition, the large amount of eukaryotic DNA collected complicates the analysis of these metagenomes at the level of assembly. Despite these complications, the subtractive analysis, i.e., what is present in PA that is not found in FL, indicated the existence of specific groups of Actinobacteria, Planctomycetes, Flavobacteria, and Proteobacteria that were not present in FL. These microbes appear to be undescribed as of yet, even by metagenomics, and justify the use of alternative approaches that target and retrieve this fraction of the marine microbiome.We have used two metagenomic approaches, direct sequencing of natural samples and sequencing after enrichment, to characterize communities of prokaryotes associated to particles. The direct sequencing of biomass collected on 5 μm filters, which in theory should collect eukaryotic cells and only prokaryotic cells forming aggregates or associated to particulate matter, accentuates the difficulties associated with evaluating particle-associated microbiomes. Our data has shown that “bona fide” free living bacteria, such as The use of culture enrichments has provided genomic data about microbes present in the sample that attach to different particulate materials. This data revealed a remarkable degree of specificity, with totally different microbes found in the different enrichments, and very little overlap despite the fact that dissolved organic carbon provided for all the enrichments, namely pyruvate, was the same. Some of the particles provided additional sources of carbon and other nutrients that could dictate some specificity. This was particularly clear in the case of chitin where classic chitinolytic microbes of the genus Vibrio were found to dominate (Hunt et al., FR, NK designed the project. ML, NK analyzed the data. JH contributed bioinformatics tools. ML, NK, and FR wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We describe the case of a 73-year-old woman with liver cirrhosis and hepatocellular carcinoma (HCC) who developed 2 distinct episodes of paroxystic atrial fibrillation (AF) each of which occurred 1 to 4 hours after iodine medium contrast-enhanced computed tomography. Sinus rhythm was restored by amiodarone therapy after the first AF episode and by electrical cardioversion after the second one. A careful clinical, biochemical, and instrumental examination showed that the patient had subclinical hyperthyroidism and moderate mitral insufficiency with mild atrial enlargement.Thus, the coexistence of both subclinical disthyroidism and of cardiac anatomical alterations may have predisposed the patient to AF that in fact occurred when exogenous iodine administration triggered a hyperthyroidism status. The HBV infection was diagnosed in 1981, when she underwent gastrectomy for complicated gastric ulcer. In February 1998, she underwent treatment with 3 million units of Interferon alpha 3 times per week per 24 weeks, without obtaining a biochemical and virological response. She was admitted to our Liver Unit in 1999. She appeared to be in satisfactory general clinical condition and reported being affected by osteoporosis . She had no history of heart, lung, kidney, metabolic, or thyroid diseases. A needle liver biopsy was performed and histological cirrhosis was diagnosed. At that time the HBV-DNA quantification showed 330,000 copies/ml  and therapy with 100 mg/die of lamivudine was therefore started. The treatment was promptly and persistently efficacious. Subsequently, the patient regularly attended the outpatient clinic of our liver unit. In February 2010, as a consequence of a slight increase of HBV-DNA levels (up to 300 IU/ml), tenofovir dipivoxil 245 mg/die therapy was introduced, replacing lamivudine.7; a total of 3 CECTs were performed without the occurrence of any side effect. In occasion of the CECT control on February 5, 2013, the patient appeared to be in good general clinical condition, the blood pressure was 130/80 mm Hg, the electrocardiogram (ECG) was normal, the heart rate was 68 beats per minutes (bpm), the blood tests were normal, HBV-DNA was undetectable. However, 4 hours after the CECT scan with iodine contrast medium administration the patient complained of palpitations, dizziness, and epigastric pain. The blood pressure was 100/60 mm Hg, radial pulse was frequent and irregular, and ECG revealed the presence of high frequency AF (120 bpm). The cardiac enzymes, evaluated every 6 hours from the onset of AF for 24 hours, were persistently within normal ranges. She was treated intravenously with amiodarone 5 mg/kg for the first hour and subsequently at the dosage of 50 mg/hour for maintenance and with subcutaneous 6000 IU/die of enoxaparin. Twenty-four hours after onset of symptoms, there was a reversion to sinus rhythm and on February 6 the patient was discharged from the hospital . Iodopride at a concentration of 370 mg/ml was the contrast medium used and it was administered intravenously at a dosage of 1.2 ml per kilogram of body weight. In December 2011, the patient underwent percutaneous radiofrequency ablation of the lesion and was subsequently followed up with periodic CECT examinations, according to AASLD practice guidelinesFour months later the patient was scheduled for a further CECT scan control. At admission on June 10, she exhibited laboratory tests—including thyroid hormone values—within normal limits, she had no symptoms, her liver disease was compensated, the blood pressure was 110/60 mm Hg, the ECG revealed a sinus rhythm with heart rate of 68 bpm. One hour after the intravenous iodide contrast medium administration, she complained about an intense chest pain without any irradiation. The blood pressure was 100/70 mm Hg, her heart rate was irregular with 150 bpm and ECG analysis identified AF. The cardiac enzyme levels were within normal ranges. Therapy with aiodarone was promptly started but this time it had no clinical effect. Therefore the patient underwent electrical cardioversion, which succeeded in reverting to sinus rhythm Figures . During 8Iodine contrast media are the most common compounds used for enhancing X-ray-based imaging methods. Their administration is responsible for an excess of exogenous free iodine in the blood and may cause thyrotoxicosis in patients with preexisting thyroid abnormalities.9 In the case of increased levels of thyroid hormones, an augmented contractile function of the myocardium has been observed, caused by upregulated synthesis of both myosin heavy chains and calcium ATPase.10 This hypercontractile state may lead to cardiac rhythm disturbances. Sinus tachycardia is the most common of such cardiac disturbances, but AF is also quite frequently observed since it occurs in 10% to 15% of patients.11Thyroid hormones have different mechanisms of action in different tissues, including vascular smooth muscle and myocardium.Although one cannot exclude that intravenous amiodarone therapy administered at the time of paroxysmal AF episodes might have contributed to thyroid dysfunction, the peculiar outcome observed in our patient can suggest that a cascade of events may have occurred depicting the following clinical scenario: a subclinical thyroid dysfunction as well as clinically silent mitral valve insufficiency and left atrium enlargement were chronically present; exogenous iodine administration triggered a hyperthyroid phase that in turn; led to AF favored by the preexisting cardiac anatomical abnormalities.In conclusion, this peculiar and interesting case suggests the advisability of investigating the possible presence of subclinical forms of thyroid and cardiac alterations in patients requiring contrast iodine administration, above all when such procedures have to be repeatedly performed as in cirrhotic patients undergoing contrast enhanced tomography for the diagnostic and therapeutic management of HCC.


PLA2G6]-related disorders), and it has management implications. © 2013 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.Back arching was reported in one of the very first patients with neurodegeneration with brain iron accumulation syndrome (NBIAs) published in 1936. However, recent reports have mainly focused on the genetic and imaging aspects of these disorders, and the phenotypic characterization of the dystonia has been lost. In evaluating patients with NBIAs in our centers, we have observed that action-induced dystonic opisthotonus is a common and characteristic feature of NBIAs. Here, we present a case series of patients with NBIAs presenting this feature demonstrated by videos. We suggest that dystonic opisthotonus could be a useful “red flag” for clinicians to suspect NBIAs, and we discuss the differential diagnosis of this feature. This would be particularly useful in identifying patients with NBIAs and no iron accumulation as yet on brain imaging  [ With the advent in genetics, a variety of complicated recessive dystonia syndromes have been identified, and the similarity in their clinical presentations makes the differential diagnosis for clinicians difficult.NBIAs cause complicated dystonia syndromes and are characterized by excessive iron deposition in the brain, particularly affecting the basal ganglia and mainly the globus pallidus. The 2 core NBIAs are the neuroaxonal dystrophies pantothenate kinase (PKAN)-associated and phospholipase A2, group IV  (PLA2G6)-associated neurodegeneration (PLAN), whereas additional disorders recently have been described.Numerous early and later case reports, in which clinical descriptions are detailed, describe neck arching and back arching in NBIAsPANK2) mutations had dystonia, including action-induced axial dystonia, but it is unknown how many patients had extensor axial dystonia20A brief review of more recent literature on NBIAs reveals that, in large series of patients that were published mostly after the identification of causative genes, although generalized, truncal and neck dystonia are described, and a more specific phenotypic description of the dystonia is largely missing.PANK2 and PLA2G6 mutations (see video segments 1–3), but not in patients with neuroferritinopathy or aceruloplasminaemia (N = 3), which is consistent with the literature, as mentioned above. Detailed clinical descriptions and imaging and genetic findings in these patients with opisthotonus have been published elsewhere but are summarized briefly in the video legend.We retrospectively evaluated all patients with NBIAs who have been seen at our center (London) the last 5 years (N = 8) and observed dystonic opisthotonus in 4 of 5 of patients with The differential diagnosis of dystonic opisthotonus includes mainly secondary dystonias,29Because Wilson's disease is a common differential diagnosis in patients with young-onset dystonia syndromes, we screened 100 patients who had symptomatic neurologic Wilson's disease who were followed in the Wilson's disease clinic  for retrocollis and opisthotonus. We reviewed the medical records and serial videos that were taken at approximately 3-month intervals over the last 7 years. Consistent with other reports,In patients diagnosed with dystonic cerebral palsy, opisthotonus has been described occasionally, but some patients with so-called cerebral palsy may have other conditions (including NBIAs)46The pathophysiologic explanation of the anatomic predilection for oromandibular and extensor truncal dystonia in these patients, as opposed to primary dystonias, remains unknown.PANK2 and PLA2G6 mutations and suggest that this feature, together with other “red flags” for NBIAs (such as severe oromandibular dystonia) should raise suspicion to test for these disorders in patients with young-onset, complicated dystonia syndromes. Hence, these patients should have appropriate imaging, which includes T2* and susceptibility-weighted imaging to look for brain iron accumulation. Phenotypic “red flags” are important for clinicians for many reasons. First, some patients with NBIAs may not initially have evidence of iron accumulation in brain imaging (as in Patients 3 and 4 presented here), and suspicion for genetic testing can be mainly guided by phenotypic clues; otherwise, misdiagnosis for many years may occur.We identified dystonic opisthotonus as a characteristic feature of NBIAs related to Video 1. Four patients with NBIAs and dystonic opisthotonus are shown. None of the patients were receiving neuroleptics before the onset of dystonic opisthotonus.Segment 1. This Indian man aged 39 years carries homozygous mutations  in the PANK2 gene. He developed decrease in visual acuity and dysarthria at age 12 years and lower limb dystonia and dystonic opisthotonus at age 14 years. On examination at age 34 years, he had reduced visual acuity, slow and hypometric vertical and horizontal saccades, generalized dystonia with prominent oromandibular dystonia, and severe dystonic opisthotonus, which was more evident while walking. Brain MRI revealed an “eye-of-the-tiger” sign.21Segment 2. This Indian woman aged 36 years (the sister of patient 1) carries the same mutation. At age 13 years, she developed visual disturbances, dysarthia, and writer's cramp on the right.Segment 3. This Pakistani man aged 21 years, the product of double consanguinity, carries homozygous c.2239C_T (p.R747W) mutations in the PLA2G6 gene.Segment 4. This Taiwanese woman aged 25 years caries a compound heterozygous mutation of the PLA2G6 gene (p.Asp331Tyr/p.Met358IlefsX). She noticed unsteady gait and easy falls at age 8 years, developed cognitive decline at age 18 years, and developed dystonia at age 22 years. Examination at age 25 years revealed retrocollis and dystonic opisthotonus induced by walking, parkinsonism, ataxic gait, intellectual impairment, and dysarthria. Brain MRI revealed cortical and cerebellar atrophy but no evidence of iron deposition on T2* sequences.22


We review here our recent work on the effects of mutations on the human phosphoglycerate kinase 1 (hPGK1), with a particular focus on thermodynamics and kinetics of protein folding and misfolding. Expression analyses and in vitro biophysical studies indicate that disease-causing mutations enhance protein aggregation propensity. We found a strong correlation among protein aggregation propensity, thermodynamic stability, cooperativity and dynamics. Comparison of folding and unfolding properties with previous reports in PGKs from other species suggests that hPGK1 is very sensitive to mutations leading to enhance protein aggregation through changes in protein folding cooperativity and the structure of the relevant denaturation transition state for aggregation. Overall, we provide a mechanistic framework for protein misfolding of hPGK1, which is insightful to develop new therapeutic strategies aimed to target native state stability and foldability in hPGK1 deficient patients.Conformational diseases are often caused by mutations, altering protein folding and stability  These mutations may affect physical properties of the protein  or the interaction of the protein along its folding process with elements of the protein homeostasis network .,45.27,45in vivo to a prompt degradation of the enzyme through the ubiquitin-proteasome pathway, mainly affecting red blood cells and the central nervous system, the former being prevented from protein synthesis, the latter being unsuited to quickly replace the enzyme. Conversely, muscle cells, which rely on an efficient protein turnover, can renew the enzyme fraction damaged by a sudden increase of body temperature, as it occurs with fever or strenuous exercises. Thus, it comes that these variants, although affected in their catalytic capacity, are active enough to sustain glycolysis, providing muscle with suitable ATP production.Mutant enzymes displaying highly reduced thermal stability, but moderately perturbed catalytic properties  show the most homogeneous correlation with hPGK1 deficient clinical phenotype . Based on crystallographic studies of the hPGK1 in the open and closed conformations, it is inferred that the mutations involve amino acids, which play a main role in preserving protein structure. All amino acids are part of the most common regular elements of the secondary structure  and participate to hydrogen/ionic interactions. In addition, I47 and L89 are involved in several hydrophobic interactions, even between them. Very interesting, L89 and A354 are substituted by a proline, a residue which is known to have a destabilizing effect on α-helical conformation and other complex effects on protein folding ,64. ConcAnother group of mutants  turn out to be heavily affected in both catalytic properties and protein stability, but one (p.T378P). Indeed, all amino acids affected by mutation have a role in preserving hPGK1 structure, sharing in β-strands  and α-helices (α-helix 7 and 13) and participating to several hydrogen/ionic interactions, mostly in both open and closed conformation ,45. ThreIn a third group of hPGK1 enzymes , the molecular properties resemble those of the WT enzyme, but intriguingly mutations are associated to heterogeneous clinical phenotypes. Thus, the genotype/phenotype relationship is quite difficult to be drawn. Very likely, amino acid substitutions are not accountable for the clinical manifestations of the carriers and the occurrence of additional genetic and/or epigenetic or other unknown factors has to be called into question for clinical phenotype explanation.E. coli at 37°C. Despite this expression system oversimplifies the natural protein homeostasis environment of human proteins, it is useful to identify and characterize folding defects of disease-causing variants, and also to evaluate its potential modulation by molecular chaperones in a simple manner [i.e., enhanced protein turnover; [Expression analyses in prokaryotes and eukaryotic cells are insightful tools to characterize protein misfolding in conformational diseases within a biologically relevant scenario ,80,81,82urnover; ,80,83). Inactivation studies have revealed a decreased stability towards thermal induced denaturation for a large fraction of the mutants causing hPGK1 deficiency . k, and its temperature dependence is described by the Arrhenius equation according to a given activation energy (Ea). DSC analyses allow compare the rate constants between hPGK1 enzymes with very different kinetic stabilities by building up Arrhenius plots used to estimate the rate constants to a given temperature  . The preperature C. These Tm value [Cp) of 9.1±0.8 kcal·mol−1·K−1. This experimental ΔCp is consistent with the global denaturation of a protein of size of hPGK1, thus indicating that irreversible denaturation of hPGK involves a large loss of tertiary structure, and also, that WT and mutant hPGK1 enzymes show similar conformations in the native and the irreversibly denatured final states [Ea on Tm values was also found for this set of enzymes, which would involve a large activation ΔCp using the same structural interpretation as for ΔCp, thus implying a denaturation transition state  as unfolded as the irreversibly denatured state. However, we proposed an alternative interpretation to this behavior based on the Hammond postulate for protein folding/unfolding [i.e., in the height of the kinetic barrier) may be explained by a more native-like structure of the denaturation transition state in the most destabilizing mutants [Hammond behavior was experimentally supported by detailed calorimetric measurements [The DSC analyses on WT and mutant enzymes have also provided insight into the denaturation mechanism and the energetic bases underlying mutation induced kinetic destabilization in hPGK1 deficiency. The denaturation enthalpies of the hPGK1 mutants are strongly dependent on the individual Tm value . This tel states . Interesnfolding , which d mutants . This Haurements , also hiurements  as well urements ,87,88.Tm (about 53 °C) and a high activation energy for irreversible thermal denaturation  to provide sufficient kinetic stability [native-like denaturation transition state [It must be noted that the high kinetic stability of WT hPGK1 at physiological temperature arises from a combination of a moderately high m and Ea . An intetability . In the tability ,88. Convon state , thus shCm and meq, which allow determine the thermodynamic stability (ΔGU) of the enzymes; (ii) thermal denaturation parameters, Ea  and Tm ; (iii) kinetic rate constants for denaturation/aggregation (from DSC experiments), for global unfolding (from urea unfolding kinetics) and for proteolysis at low/high protease concentrations limits . The comparison between the different sets of rate constants allows to obtain information on the denaturation and proteolysis kinetic mechanisms (see below). The phenomenological two-state model (N→F) used to describe thermal aggregation and denaturation of hPGK1 enzymes  and Figu does not−1 for WT hPGK1, which is in the same range of the thermodynamic stability found for PGKs from E. coli, yeast and muscle horse [Urea denaturation of WT hPGK1 shows a single transition using different structural probes ,46 consile horse ,15. We mle horse ,16,23. k is equal to K·kp; here, the kinetics of aggregation depends on the thermodynamic stability  and on the intrinsic aggregation rate ; B). Aggregation (U→F) is faster than refolding (U→N), and thus, the overall aggregation rate constant approaches the unfolding rate constant for N→U step. Indeed, the rate constants for global unfolding determined by urea denaturation and extrapolated to the absence of denaturant show that global unfolding is much faster . Equilibrium denaturation experiments also support that aggregation kinetics correlate well with a decreased thermodynamic stability of the native state, further supporting that scenario A describes well the increased aggregation propensity of hPGK1 mutants by (at least) thermodynamic destabilization of the protein [There are two limiting situations in the N↔U→F mechanism proposed above ,89: A) t protein . resembles the globally unfolded state [Alternatively, we have used proteolysis to obtain further insight on the mechanism by which hPGK1 mutants reduce the kinetic stability of the native state. Kinetics of proteolysis can be studied using a similar three-state kinetic mechanism to the one proposed for aggregation kinetics hPGK1 with a small conceptual modification: the state undergoing the irreversible denaturation is a protease sensitive state (named X instead of U), while the final state F is the degraded protein. Accordingly, at low protease concentrations, proteolysis is rate-limiting (scenario A) and at high protease concentrations, proteolysis kinetics approaches to kinetics of formation of the protease sensitive state (scenario B) ,91. We hed state . At low ed state ). At higed state ). E. coli PGK is 3–4 orders of magnitude more kinetically resistant towards unfolding and proteolysis than hPGK1 and yeast PGK, despite their similar thermodynamic stabilities [E. coli PGK, the kinetically sensitive state is the global unfolded state, while in yeast PGK is a partially folded state [bilities ,46. Thisbilities ,36. In hed state ,36. It hed state . i.e., the equilibrium m-value; E. coli show remarkably similar equilibrium m values  consrativity ,93. Low rativity ,95,96, erativity  and Figu−1, while global unfolding kinetics shows a kinetic destabilization of less than 1 kcal·mol−1 . Comprehensive studies of all these factors will help to understand loss-of-function in hPGK1 deficiency, as well as individual differences in the protein homeostasis network [iscosity ,99,100. iscosity ,101. Hownditions . So, it  effects . These c effects . Moreove effects ,99. All  network  which main vitro at physiological temperatures (ongoing research). Detailed analyses of protein stability also show that thermodynamic destabilization of the native state may play a role in aggregation/degradation of mutant enzymes . Interestingly, the p.T378P mutant also seems to slow down refolding kinetics [in vitro and in E. coli expression studies. Thus, it is reasonable that targeting the coordinated action of molecular chaperones on hPGK1 folding intermediates may also allow increase the protein folding efficiency of these disease causing mutants (Molecular chaperones are known to help proteins to fold and to prevent aggregation of partially folded states . Our unfin vitro . Howeverin vitro  and ongokinetics . The hig mutants . Phenoty