A novel combination approach to effectively reduce inflammation and neurodegeneration in multiple sclerosis models.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated attacks on the central nervous system (CNS), resulting in demyelination and recurring T-cell responses. Unfortunately, there is no cure for it. Current therapies that target immunomodulation and/or immunosuppression show only modest beneficial effects, have many side effects, and do not block neurodegeneration or progression of the disease. Since neurodegeneration and in particular axonal degeneration is implicated in disability in progressive MS, development of novel therapeutic strategies to attenuate the neurodegenerative processes is imperative. This study aims to develop new safe and efficacious treatments that address both the inflammatory and neurodegenerative aspects of MS using its animal model, experimental allergic encephalomyelitis (EAE). In EAE, the cysteine protease calpain is upregulated in CNS tissue, and its activity correlates with neurodegeneration. Our immunologic studies on MS have indicated that increased calpain activity promotes pro-inflammatory T helper (Th)1 cells and the severity of the disease in EAE, suggesting that calpain inhibition could be a novel target to combat neurodegeneration in MS/EAE. While calpain inhibition by SNJ1945 reduced disease severity, treatment of EAE animals with a novel protease-resistant altered small peptide ligand (3aza-APL) that mimic myelin basic protein (MBP), also decreased the incidence of EAE, disease severity, infiltration of inflammatory cells, and protected myelin. A reduction in inflammatory T-cells with an increase in Tregs and myeloid suppressor cells is also found in EAE mice treated with SNJ1945 and 3aza-APL. Thus, a novel combination strategy was tested in chronic EAE mouse model in B10 mice which showed multiple pathological mechanisms could be addressed by simultaneous treatment with calpain inhibitor SNJ1945 and protease-resistant 3aza-APL to achieve a stronger therapeutic effect.

Renal transplant recipients undergoing endovascular abdominal aortic aneurysm repair have increased risk of perioperative acute kidney injury but no difference in late mortality.

OBJECTIVE: Renal transplant is associated with substantial survival advantage in patients with end-stage renal disease. However, little is known about the outcomes of renal transplant recipients (RTRs) after endovascular abdominal aortic aneurysm repair (EVAR). This study aimed to study the effect of renal transplant on perioperative outcomes and long-term survival after elective infrarenal EVAR. METHODS: The Vascular Quality Initiative database was queried for all patients undergoing elective EVAR from 2003 to 2021. Functioning RTRs were compared with non-renal transplant recipients without a diagnosis of end-stage renal disease (non-RTRs). The outcomes included 30-day mortality, acute kidney injury (AKI), new renal failure requiring renal replacement therapy (RRT), endoleak, aortic-related reintervention, major adverse cardiac events, and 5-year survival. A logistic regression analysis was used to assess the association between RTRs and perioperative outcomes. RESULTS: Of 60,522 patients undergoing elective EVAR, 180 (0.3%) were RTRs. RTRs were younger (median, 71 years vs 74.5 years; P < .001), with higher incidence of hypertension (92% vs 84%; P = .004) and diabetes (29% vs 21%; P = .005). RTRs had higher median preoperative serum creatinine (1.3 mg/dL vs 1.0 mg/dL; P < .001) and lower estimated glomerular filtration rate (51.6 mL/min vs 69.4 mL/min; P < .001). There was no difference in the abdominal aortic aneurysm diameter and incidence of concurrent iliac aneurysms. Procedurally, RTRs were more likely to undergo general anesthesia with lower amount of contrast used (median, 68.6 mL vs 94.8 ml; P < .001) and higher crystalloid infusion (median, 1700 mL vs 1500 mL; P = .039), but no difference was observed in the incidence of open conversion, endoleak, operative time, and blood loss. Postoperatively, RTRs experienced a higher rate of AKI (9.4% vs 2.7%; P < .001), but the need for new RRT was similar (1.1% vs 0.4%; P = .15). There was no difference in the rates of postoperative mortality, aortic-related reintervention, and major adverse cardiac events. After adjustment for potential confounders, RTRs remained associated with increased odds of postoperative AKI (odds ratio, 3.33; 95% confidence interval, 1.93-5.76; P < .001) but had no association with other postoperative complications. A subgroup analysis identified that diabetes (odds ratio, 4.21; 95% confidence interval, 1.17-15.14; P = .02) is associated with increased odds of postoperative AKI among RTRs. At 5 years, the overall survival rates were similar (83.4% vs 80%; log-rank P = .235). CONCLUSIONS: Among patients undergoing elective infrarenal EVAR, RTRs were independently associated with increased odds of postoperative AKI, without increased postoperative renal failure requiring RRT, mortality, endoleak, aortic-related reintervention, or major adverse cardiac events. Furthermore, 5-year survival was similar. As such, while EVAR may confer comparable benefits and technical success perioperatively, RTRs should have aggressive and maximally optimized renal protection to mitigate the risk of postoperative AKI.

Improving Liver Transplant Outcomes for Hepatitis C Virus Hepatocellular Carcinoma in the Direct-Acting Antiviral Therapy Era.

BACKGROUND: Direct-acting antiviral (DAA) therapy has transformed the outcomes of liver transplant (LT) with hepatitis C virus (HCV). This study aimed to analyze the effects of DAA treatment for HCV-associated hepatocellular carcinoma (HCC) in LT. METHODS: We included patients confirmed with HCC on explant, analyzed data from United Network for Organ Sharing, and defined the pre-DAA era (2012-2013) and DAA era (2014-2016). RESULTS: HCV-associated HCC cases totaled 4778 (62%) during the study period. In the DAA era, the median recipient age was older and the median days on the waiting list were longer. For the donor, median age, body mass index, and the rate of HCV significantly increased in the DAA era. In pathology, the median largest tumor size was significantly higher; however, the rate of completed tumor necrosis was significant higher in the DAA era. The 3-year graft/patient survival had significantly improved in the DAA era. In multivariable analysis, the DAA era (hazard ratio, 0.79; 95% confidence interval, 0.68-0.91) had significantly affected the 3-year graft survival. CONCLUSIONS: DAA has a significant beneficial effect on LT. In the DAA era, graft survival for HCV-associated HCC has been significantly improving.

Retrospective Review of Trauma ICU Patients With and Without Palliative Care Intervention.

BACKGROUND: Older trauma patients present with poor preinjury functional status and more comorbidities. Advances in care have increased the chance of survival from previously fatal injuries with many left debilitated with chronic critical illness and severe disability. Palliative care (PC) is ideally suited to address the goals of care and symptom management in this critically ill population. A retrospective chart review was done to identify the impact of PC consults on hospital length of stay (LOS), ICU LOS, and surgical decisions. STUDY DESIGN: A Level 1 Trauma Center Registry was used to identify adult patients who were provided PC consultation in a selected 3-year time period. These PC patients were matched with non-PC trauma patients on the basis of age, sex, race, Glasgow Coma Scale, and Injury Severity Score. Chi-square tests and Student's t-tests were used to analyze categorical and continuous variables, respectively. Any p value >0.05 was considered statistically significant. RESULTS: PC patients were less likely to receive a percutaneous endoscopic gastric tube or tracheostomy. PC patients spent less time on ventilator support, spent less time in the ICU, and had a shorter hospital stay. PC consultation was requested 16.48 days into the patient's hospital stay. Approximately 82% of consults were to assist with goals of care. CONCLUSION: Specialist PC team involvement in the care of the trauma ICU patients may have a beneficial impact on hospital LOS, ICU LOS, and surgical care rendered. Earlier consultation during hospitalization may lead to higher rates of goal-directed care and improved patient satisfaction.

Disparate access to breast cancer screening and treatment.

BACKGROUND: Barriers to breast cancer screening remain despite Medicaid expansion for preventive screening tests and implementation of patient navigation programs under the Affordable Care Act. Women from underserved communities experience disproportionately low rates of screening mammography. This study compares barriers to breast cancer screening among women at an inner-city safety-net center (City) and those at a suburban county medical center (County). Inner city and suburban county medical centers' initiatives were studied to compare outcomes of breast cancer screening and factors that influence access to care. METHODS: Women 40 years of age or older delinquent in breast cancer screening were offered patient navigation services between October 2014 and September 2019. Four different screening time-to-event intervals were investigated: time from patient navigation acceptance to screening mammography, to diagnostic mammography, to biopsy, and overall screening completion time. Barriers to complete breast cancer screening between the two centers were compared. RESULTS: Women from lowest income quartiles took significantly longer to complete breast cancer screening when compared to women from higher income quartiles when a barrier was present, regardless of barrier type and center. Transportation was a major barrier to screening mammography completion, while fear was the major barrier to abnormal screening work up. CONCLUSION: Disparity in breast cancer screening and management persists despite implementation of a patient navigation program. In the presence of a barrier, women from the lowest income quartiles have prolonged breast cancer screening completion time regardless of center or barrier type. Women who experience fear have longest screening time completion. Future directions aim to increase resource allocation to ameliorate wait times in overburdened safety-net hospitals as well as advanced training for patient navigators to alleviate women's fears.

Ruthenium Metallotherapeutics: Novel Approaches to Combatting Parasitic Infections.

Human parasitic infections cause a combined global mortality rate of over one million people per annum and represent some of the most challenging diseases for medical intervention. Current chemotherapeutic strategies often require prolonged treatment, coupled with subsequent drug-induced cytotoxic morbidity to the host, while resistance generation is also a major concern. Metals have been used extensively throughout the history of medicine, with more recent applications as anticancer and antimicrobial agents. Ruthenium metallotherapeutic antiparasitic agents are highly effective at targeting a range of key parasites, including the causative agents of malaria, trypanosomiasis, leishmaniasis, amoebiasis, toxoplasmosis and other orphan diseases, while demonstrating lower cytotoxicity profiles than current treatment strategies. Generally, such compounds also demonstrate activity against multiple cellular target sites within parasites, including inhibition of enzyme function, cell membrane perturbation, and alterations to metabolic pathways, therefore reducing the opportunity for resistance generation. This review provides a comprehensive and subjective analysis of the rapidly developing area of ruthenium metal- based antiparasitic chemotherapeutics, in the context of rational drug design and potential clinical approaches to combatting human parasitic infections.

Pelvic fractures in severely injured elderly: a double-adjustment propensity score matched analysis from a level I trauma center.

PURPOSE: Pelvic trauma has increased risk of mortality in the elderly. Our study aimed to analyze the impact of the additional burden of pelvic fractures in severely injured elderly. METHODS: This is a retrospective analysis of a prospectively maintained trauma registry from 2012 to 2018 at an American College of Surgeons (ACS) verified Level I Trauma Center. Trauma patients aged ≥ 65 years with ISS ≥ 16 and AIS severity score ≥ 3 in at least two body regions were divided in two groups: group I, consisted of elderly polytrauma patients without pelvic fractures, and group II elderly who had concomitant pelvic fractures. We used a double-adjustment method using propensity score matching (PSM) with subsequent covariate adjustment to minimize the effect of confounding factors, and give unbiased estimation of the impact of pelvic fractures. Balance assessment was conducted by computing absolute standardized mean differences (ASMDs) and ASMD < 0.10 reflects good balance between groups. RESULTS: Of 12,774 patients admitted during this time, 411 (3.2%) elderly with a mean age of 77.75 ± 8.32 years met the inclusion criteria. Of this cohort, only 92 patients (22.4%) had pelvic fractures. Females outnumbered males (55 vs. 45%). Comparing characteristics of group I and group II using ASMDs, pelvic trauma patients were more likely to have higher systolic blood pressure (SBP), head injuries, lower extremity injuries, anticoagulant therapy, and cirrhosis. Fewer variables differed significantly after matching. We observed few instances of worse outcomes associated with pelvic trauma using PSM with and without covariate adjustment. Crude PSM without covariate adjustment, showed a significantly higher rate of deep vein thrombosis (DVT) for pelvic trauma (p < 0.001). Crude PSM also showed a significantly higher rate of ventilator-associated pneumonia (VAP) in group II (p = 0.006). PSM with covariate adjustment did not confirm differences on these outcomes. PSM both without and with covariate adjustment found lower ventilator days and ICU length of stay among patients with pelvic trauma. No significant differences were seen on 12 outcomes: death, acute kidney injury (AKI), acute respiratory distress syndrome (ARDS), cardiac arrest with cardiopulmonary resuscitation (CPR), myocardial infarction (MI), pulmonary embolism (PE), unplanned intubation, unplanned admission to intensive care unit (ICU), catheter-associated urinary tract infection (CAUTI), and hospital length of stay. CONCLUSIONS: At a Level I Trauma Center the additional burden of pelvic fractures in seriously injured elderly did not translate into higher mortality. PSM without covariate adjustment suggests worse rates among pelvic trauma patients for DVT and VAP but covariate adjustment removed statistical significance for both outcomes. Pelvic trauma patients had shorter time on ventilator and in the ICU. Whether similar analytic methods applied to patients from larger data sources would produce similar findings remains to be seen.

Practice patterns and outcomes for pediatric ANCA vasculitis transplant recipients in a national cohort.

BACKGROUND: Anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis is a rare condition in pediatric patients. Little is known about practice patterns and outcomes of pediatric transplant patients. The purpose of our study was to examine differences in patient characteristics, immunosuppression, and long-term graft outcomes between ANCA and non-ANCA vasculitis recipients. METHODS: We used the Scientific Registry of Transplant Recipients to evaluate pediatric ANCA vasculitis recipients between the ages of 1 and 22 years old from 1991 to 2017 and compared them to non-ANCA vasculitis patients during the same time cohort in the USA. RESULTS: A total of 26,431 transplant recipients were identified, of these, 337 with ANCA vasculitis. Mean 1-year eGFR was 62.46 and 64.92 ml/min/1.73 m2 (p = 0.002), and mean 5-year eGFR was 57.95 and 59.38 ml/min/1.73 m2 (p = 0.18) between the non-ANCA and ANCA groups, respectively. Five-year graft survival was similar in both groups (non-ANCA 75.5 vs. ANCA 78.6%; p = 0.19). Of those with graft loss within the ANCA group, only 0.6% was secondary to disease recurrence (p = 0.14). CONCLUSIONS: Kidney transplant is a safe treatment modality for children with ANCA-related kidney failure. ANCA patients have comparable graft survival when compared to the general transplant population with a low risk of recurrence. Thymoglobulin was used in a higher proportion within the ANCA group compared to the non-ANCA group. Tacrolimus, mycophenolate mofetil/mycophenolic acid, and steroids were the predominant maintenance immunosuppression used in both groups. A higher resolution version of the Graphical abstract is available as Supplementary information.

Prophylactic sac outflow vessel embolization is associated with improved sac regression in patients undergoing endovascular aortic aneurysm repair.

OBJECTIVE: Type II endoleaks (T2Es), often identified after endovascular aneurysm repair (EVAR), have been associated with late endograft failure and secondary rupture. The number and size of the patent aortic aneurysm sac outflow vessels (ie, the inferior mesenteric, lumbar, and accessory renal arteries) have been implicated as known risk factors for persistent T2Es. Given the technical challenges associated with post-EVAR embolization, prophylactic embolization of aortic aneurysm sac outflow vessels has been advocated to prevent T2Es; however, the evidence available at present is limited. We sought to examine the effects of concomitant prophylactic aortic aneurysm sac outflow vessel embolization in patients undergoing EVAR. METHODS: Patients aged ≥18 years included in the Society for Vascular Surgery Vascular Quality Initiative database who had undergone elective EVAR for intact aneurysms between January 2009 and November 2020 were included in the present study. Patients with a history of prior aortic repair and those without available follow-up data were excluded. The patient demographics, operative characteristics, and outcomes were analyzed by group: EVAR alone vs EVAR with prophylactic sac outflow vessel embolization (emboEVAR). The outcomes of interest were the in-hospital postoperative complication rates, incidence of aneurysmal sac regression (≥5 mm) and T2Es, and reintervention rates during follow-up. RESULTS: A total of 15,060 patients were included. Of these patients, 272 had undergone emboEVAR and 14,788 had undergone EVAR alone. No significant differences were found between the two groups in age, comorbidities, or anatomic characteristics, including the mean maximum preoperative aortic diameter (5.5 vs 5.6 cm; P = .48). emboEVAR was associated with significantly longer procedural times (148 vs 124 minutes; P < .0001), prolonged fluoroscopy times (32 vs 23 minutes; P < .0001), increased contrast use (105 vs 91 mL; P < .0001), without a significant reduction in T2Es at case completion (17.7% vs 16.3%; P = .54). The incidence of postoperative complications (3.7% vs 4.6%; P = .56), index hospitalization reintervention rates (0.7% vs 1.3%; P = .59), length of stay (1.8 vs 2 days; P = .75), and 30-day mortality (0% vs 0%; P = 1.00) were similar between the two groups. At mid-term follow-up (14.6 ± 6.2 months), the emboEVAR group had a significantly greater mean reduction in the maximum aortic diameter (0.69 vs 0.54 cm; P = .006), with a greater proportion experiencing sac regression of ≥5 mm (53.5% vs 48.7%). The reintervention rates were similar between the two groups. On multivariable analysis, prophylactic aortic aneurysm sac outflow vessel embolization (odds ratio, 1.34; 95% confidence interval, 1.04-1.74; P = .024) was a significant independent predictor of sac regression. CONCLUSIONS: Prophylactic sac outflow vessel embolization can be performed safely for patients with intact aortic aneurysms undergoing elective EVAR without significant associated perioperative morbidity or mortality. emboEVAR was associated with significant sac regression compared with EVAR alone at mid-term follow-up. Although no decrease was found in the incidence of T2Es, this technique shows promise, and future efforts should focus on identifying a subset of aneurysm and outflow branch characteristics that will benefit from concomitant selective vs complete prophylactic sac outflow vessel embolization.

Natural Antimicrobial Nano Composite Fibres Manufactured from a Combination of Alginate and Oregano Essential Oil.

Alginate is a linear biodegradable polysaccharide polymer, which is bio-renewable and widely used for various biomedical applications. For the next generation of medical textiles, alginate nanofibres are desirable for their use in wound dressings that are biocompatible, sustainable, and abundantly available. This study has developed a unique manufacturing process for producing alginate nanofibres with exceptional antimicrobial properties of oregano essential oil (OEO) as a natural antimicrobial agent. OEO with varying degrees of concentration was incorporated in an aqueous alginate solution. Appropriate materials and electrospinning process parameter selection allowed us to manufacture alginate fibres with a range of diameters between 38 and 105 nm. A unique crosslinking process for alginate nanofibres using extended water soaking was developed. Mechanical characterisation using micro-mechanical testing of nonwoven electrospun alginate/oregano composite nanofibres revealed that it was durable. An extensive antimicrobial study was carried out on alginate/oregano composite nanofibres using a range of Gram-positive (methicillin-resistant Staphylococcus aureus (MRSA) and Listeria monocytogenes) and Gram-negative bacteria (Klebsiella pneumoniae and Salmonella enterica), which are common wound and food pathogens. The results indicated that increasing the concentration of OEO from 2 to 3 wt % showed improved antimicrobial activity against all pathogens, and activity was significantly improved against MRSA compared to a non-alginate-based control disk containing OEO. Therefore, our research suggests that all-natural alginate/oregano nanofibre composite textiles offer a new generation of medical textiles for advanced wound dressing technology as well as for food packaging applications.

Breast Cancer Screening in Inner City and County Populations: A Tale of Two Centers.

BACKGROUND: New York's statewide "Get Screened, No Excuses" campaign has been one of the nation's most aggressive actions to improve access to breast cancer screening. Inner city and suburban county medical centers' initiatives were studied to compare outcomes of breast cancer screening and factors that influence access to care. METHODS: Women delinquent in breast cancer screening one year or greater were offered patient navigator services to aid in timely breast cancer screening. Time-to-event completion rates among different stages of breast cancer screening stages in City and County women were compared. Time-to-event completion rates among different stages of breast cancer screening stages. RESULTS: 2505 women aged ≥40 years accepted PN services. Mean (SD) age of patients was 56.2 (10) years. The mean (SD) age of those who completed breast screening vs. those who did not was 56.8 (10) and 52.5 (.9) years, respectively (P < .01). The rates of screening completion during physical examination, mammography and biopsy stages were 74%, 78% and 100% in City vs. 98%, 85% and 100% in County, respectively (P < .001). Screening phase was the significant predictor of time to completion for breast cancer screening in Cox regression analysis. Over 85% of women completed the breast cancer screening, 74% in City and 97.6% in County (P < .001). DISCUSSION: Screening phase is an important predictor of time-to complete breast cancer screening. Center location served as the effect modifier of the relationship. The rate of completing the screening was significantly higher and faster among Suburban County compared to Inner City women.

The Microbiology of Ruthenium Complexes.

Ruthenium is seldom mentioned in microbiology texts, due to the fact that this metal has no known, essential roles in biological systems, nor is it generally considered toxic. Since the fortuitous discovery of cisplatin, first as an antimicrobial agent and then later employed widely as an anticancer agent, complexes of other platinum group metals, such as ruthenium, have attracted interest for their medicinal properties. Here, we review at length how ruthenium complexes have been investigated as potential antimicrobial, antiparasitic and chemotherapeutic agents, in addition to their long and well-established roles as biological stains and inhibitors of calcium channels. Ruthenium complexes are also employed in a surprising number of biotechnological roles. It is in the employment of ruthenium complexes as antimicrobial agents and alternatives or adjuvants to more traditional antibiotics, that we expect to see the most striking developments in the future. Such novel contributions from organometallic chemistry are undoubtedly sorely needed to address the antimicrobial resistance crisis and the slow appearance on the market of new antibiotics.

Estrogen or estrogen receptor agonist inhibits lipopolysaccharide induced microglial activation and death.

Inflammation is an important pathogenic mechanism in many neurodegenerative disorders. Activated microglia play a pivotal role in releasing pro-inflammatory factors including interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2 (COX-2) for inducing inflammation. While microglia mediated inflammation is essential in maintaining CNS homeostasis, chronic inflammation results in activation of proteases for cell death. Here, we examined the effect of PPT (estrogen receptor α agonist), DPN (estrogen receptor ß agonist), and estrogen on rat primary microglia following exposure to lipopolysaccharide (LPS). Exposure of microglia to LPS (200 ng/ml) for 24 h induced cell death. After LPS toxicity for 15 min, microglia were treated with 25 nM PPT, 25 nM DPN, or 100 nM estrogen that prevented cell death by attenuating the release of IL-1α, IL-1ß, TNF-α, and COX-2. Treatment of cells with 100 nM fulvestrant (estrogen receptor antagonist) prior to addition of PPT, DPN, or estrogen significantly decreased their ability to prevent cell death, indicating involvement of estrogen receptor (ER) in providing PPT, DPN, or estrogen mediated cytoprotection. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses showed alterations in mRNA expression of Bax, Bcl-2, calpain, and calpastatin during apoptosis. We also examined mRNA expression of ERß and ERα following exposure of microglia to LPS and subsequent treatment with PPT, DPN, or estrogen. We found that estrogen or estrogen receptor agonists upregulated expression of ERs. Overall, results indicate that estrogen receptor agonist or estrogen uses a receptor mediated pathway to protect microglia from LPS toxicity.

Calpeptin attenuated inflammation, cell death, and axonal damage in animal model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose dependently with the calpain inhibitor calpeptin (50-250 microg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased, with down-regulation of proapoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of proapoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.

Immunolocalization of mitoKATP subunits in human osteoblast-like cells.

The mitochondrial ATP-dependent K channel (mitoKATP) has been shown to play a role in cellular protection against apoptosis, or programmed cell death. This channel has been identified and characterized in a number of cell and tissue types but to date the possible existence of mitoKATP in osteoblastic cells has not been investigated. The aim of this investigation was to establish whether the mitochondria of human osteosarcoma-derived osteoblasts (SaOS-2 cells) contain the putative mitoKATP subunits Kir6.1 and Kir6.2. Ultrathin sections of SaOS-2 cells were prepared for transmission electron microscopy using an adaptation of the Tokuyasu method, and immunolabelled using goat anti-Kir6.1 or anti-Kir6.2 antisera as the primary label, and a 10nm colloidal gold-conjugated donkey anti-goat secondary antibody. The suitability of the antisera and the immunostaining protocol were confirmed by using a sample of rat cardiac muscle as a positive control. Ultrastructural analysis revealed that SaOS-2 cells contain Kir6.2 but not Kir6.1, and that Kir6.2 is present in the mitochondria, but in extremely low abundance. These findings suggest that human osteoblast-like cells might contain mitoKATP channels in which Kir6.2 is the pore-forming subunit, although it appears that these channels are likely to be present in extremely low abundance.

N-(4-Hydroxyphenyl) retinamide potentiated paclitaxel for cell cycle arrest and apoptosis in glioblastoma C6 and RG2 cells.

Glioblastoma grows aggressively due to its ability to maintain abnormally high potentials for cell proliferation. The present study examines the synergistic actions of N-(4-hydroxyphenyl) retinamide (4-HPR) and paclitaxel (PTX) to control the growth of rat glioblastoma C6 and RG2 cell lines. 4-HPR induced astrocytic differentiation that was accompanied by increased expression of the tight junction protein e-cadherin and sustained down regulation of Id2 (member of inhibitor of differentiation family), catalytic subunit of rat telomerase reverse transcriptase (rTERT), and proliferating cell nuclear antigen (PCNA). Flow cytometric analysis showed that the microtubule stabilizer PTX caused cell cycle deregulation due to G2/M arrest. This in turn could alter the fate of kinetochore-spindle tube dynamics thereby halting cell cycle progression. An interesting observation was the induction of G1/S arrest by a combination of 4-HPR and PTX, altering the G2/M arrest induced by PTX alone. This was further ratified by the upregulation of tumor suppressor protein retinoblastoma, which repressed the expression of the key signaling moieties to induce G1/S arrest. Collectively, the combination of 4-HPR and PTX diminished the survival factors (e.g., rTERT, PCNA, and Bcl-2) to make glioblastoma cells highly prone to apoptosis with activation of cysteine proteases (e.g., calpain, cathepsins, caspase-8, caspase-3). Hence, the combination of 4-HPR and PTX can be considered as an effective therapeutic strategy for controlling the growth of heterogeneous glioblastoma cell populations.

Involvement of calpain in the process of Jurkat T cell chemotaxis.

Massive T cell infiltration into the central nervous system is a hallmark of multiple sclerosis (MS) and its rodent model experimental autoimmune encephalomyelitis (EAE), resulting in the induction of many of the pathophysiological events that lead to neuroinflammation and neurodegeneration. Thus, blocking T cell migration into the central nervous system may reduce disease severity in MS and EAE. One potential target for reducing T cell migration is inhibition of the Ca(2+)-activated neutral protease calpain. Previous studies in other cell types have demonstrated that migration is reduced by incubation of cells with calpain inhibitors. Thus, we hypothesize that calpain inhibition will reduce migration of T cells in response to and toward the chemokine CCL2. To test this hypothesis, the intracellular free Ca(2+) levels in Jurkat E6-1 T cells was first measured by the fura-2 assay to assess whether the intracellular ion environment would support calpain activation. The intracellular free Ca(2+) levels were found to increase in response to CCL2. The cells were next treated with the calpain inhibitor calpeptin in a multiwelled Boyden chamber with CCL2 used as the chemoattractant. These studies demonstrate that inhibition of calpain with its inhibitor calpeptin produces a dose-dependent inhibition of chemotaxis. Calpain activity, as measured by live cell imaging, was also increased in response to CCL2, providing further evidence of its involvement in the process of chemotaxis and migration. These studies provide evidence for the involvement of calpain in the mechanisms of chemotaxis and warrants further exploration in MS patient and EAE animal samples.

Congenital insensitivity to pain--review and report of a case with dental implications.

Pain is a protective mechanism for the body. Absence of pain is a symptom in several disorders, both congenital and acquired. The congenital types are present at birth and affect the number and distribution of types of nerve fibers. At present, 5 types of hereditary sensory and autonomic neuropathies have been identified. The various disorders within this group are classified according to the different patterns of sensory and autonomic dysfunction and peripheral neuropathy and the presence of additional clinical features such as learning disability. However, the field is currently moving away from classification based on clinical presentation toward classification based on underlying genetic abnormality. In the absence of pain, patients are at risk of late presentation with illnesses or injuries, and have an increased incidence of traumatic injury. Self-mutilation is an almost invariable feature of these disorders. We report the case of a patient with congenital insensitivity to pain that presented with self-mutilation injuries to his hands and oral tissues caused by biting. The severe nature of these injuries necessitated serial extraction of his primary teeth soon after eruption, which led to a cessation of the problem. The mutilation has not returned following the eruption of the first of his permanent teeth, suggesting that he has learned not to bite himself, even though to do so causes him no discomfort.

Initial sequencing and comparative analysis of the mouse genome.

The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism.