Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. METHOD AND RESULTS: The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. CONCLUSIONS: Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development.

A retrospective study on epidemiology of hypoglycemia in Emergency Department.

BACKGROUND: Hypoglycemia is one among the leading causes for Emergency Department (ED) visits and is the most common and easily preventable endocrine emergency. This study is aimed at assessing the incidence and elucidating the underlying causes of hypoglycemia. MATERIALS AND METHODS: A retrospective, observational study which included patients registering in ED with a finger prick blood glucose ≤60 mg/dl at the time of arrival. All patients aged above 15 years with the above inclusion criteria during the period of August 2010 to July 2013 were selected. The study group was categorized based on diabetic status into diabetic and nondiabetic groups. RESULTS: A total of 1196 hypoglycemic episodes encountered at the ED during the study period were included, and of which 772 with complete data were analyzed. Underlying causes for hypoglycemia in the diabetic group (535) mainly included medication related 320 (59.81%), infections 108 (20.19%), and chronic kidney disease 61 (11.40%). Common underlying causes of hypoglycemia in nondiabetic group (237, 30.69%) included infections 107 (45.15%), acute/chronic liver disease 42 (17.72%), and malignancies 22 (9.28%). Among diabetic subjects on antidiabetic medications (n = 320), distribution over 24 h duration clearly reported two peaks at 8th and 21st h. The incidence of hypoglycemia and death per 1000 ED visits were 16.41 and 0.73 in 2011, 16.19 and 0.78 in 2012, 17.20 and 1.22 in 2013 with an average of 16.51 and 0.91, respectively. CONCLUSION: Bimodal distribution with peaks in incidences of hypoglycemic attacks at 8th and 21st h based on hourly distribution in a day can be correlated with the times just before next meal. None of the patients should leave ED without proper evaluation of the etiology of hypoglycemia and the problem should be addressed at each individual level. Increasing incidence of death over the years is alarming, and further studies are needed to conclude the root cause.

Fluorescently labeled bacteria provide insight on post-mortem microbial transmigration.

Microbially mediated mechanisms of human decomposition begin immediately after death, and are a driving force for the conversion of a once living organism to a resource of energy and nutrients. Little is known about post-mortem microbiology in cadavers, particularly the community structure of microflora residing within the cadaver and the dynamics of these communities during decomposition. Recent work suggests these bacterial communities undergo taxa turnover and shifts in community composition throughout the post-mortem interval. In this paper we describe how the microbiome of a living host changes and transmigrates within the body after death thus linking the microbiome of a living individual to post-mortem microbiome changes. These differences in the human post-mortem from the ante-mortem microbiome have demonstrated promise as evidence in death investigations. We investigated the post-mortem structure and function dynamics of Staphylococcus aureus and Clostridium perfringens after intranasal inoculation in the animal model Mus musculus L. (mouse) to identify how transmigration of bacterial species can potentially aid in post-mortem interval estimations. S. aureus was tracked using in vivo and in vitro imaging to determine colonization routes associated with different physiological events of host decomposition, while C. perfringens was tracked using culture-based techniques. Samples were collected at discrete time intervals associated with various physiological events and host decomposition beginning at 1h and ending at 60 days post-mortem. Results suggest that S. aureus reaches its highest concentration at 5-7 days post-mortem then begins to rapidly decrease and is undetectable by culture on day 30. The ability to track these organisms as they move in to once considered sterile space may be useful for sampling during autopsy to aid in determining post-mortem interval range estimations, cause of death, and origins associated with the geographic location of human remains during death investigations.

Phase 1 study of epigenetic priming with decitabine prior to standard induction chemotherapy for patients with AML.

We conducted an open-label phase 1 study exploring the feasibility, safety, and biologic activity of epigenetic priming with decitabine before standard induction chemotherapy in patients with less-than-favorable risk of acute myelogenous leukemia (AML). We directly compared the clinical and DNA-hypomethylating activity of decitabine delivered at 20 mg/m² by either a 1-hour infusion (Arm A) or a continuous infusion (Arm B) for 3, 5, or 7 days before a single, standard induction with infusional cytarabine (100 mg/m² for 7 days) and daunorubicin (60 mg/m² × 3 doses). Toxicity was similar to that of standard induction chemotherapy alone. Although we did not identify a maximum tolerated dose, there was more gastro-intestinal toxicity with 7 days of decitabine priming. Decitabine induced DNA hypomethylation at all dose levels and there was a trend toward greater hypomethylation in CD34(+) bone marrow cells when decitabine was delivered by a short pulse (Arm A). Twenty-seven subjects (90%) responded to therapy: 17 with complete remission (57%) and 10 with partial remission (33%). Of the patients with partial remission to protocol treatment, 8 achieved remission to their next therapy, bringing the overall complete remission rate to 83%. We conclude that epigenetic priming of intensive chemotherapy can be safely delivered in an attempt to improve response rates. This trial was registered at www.clinicaltrials.gov as NCT00538876.

Limb salvage surgery complimented by customised mega prostheses for malignant fibrous histiocytomas of bone.

PURPOSE: To assess functional and oncological outcomes of patients with malignant fibrous histiocytomas of bone, after limb salvage surgery complimented by a customised prosthesis. METHODS: Between May 1991 and December 2002, 15 men and 5 women (mean age, 42 years) with histologically proven malignant fibrous histiocytoma of bone underwent treatment involving limb salvage surgery complimented by a customised mega prosthesis. Most of the tumours were stage II according to the Enneking system, and located around the knee. Wide resection margins were achieved in 18 patients. RESULTS: Following a mean follow-up of 58 months, 4 patients underwent amputation for local recurrence and 5 died of the disease. Two patients had prosthesis fractures; revision of the prosthesis was carried out in one. The functional result was excellent in 5 and good in 9 patients. The Kaplan-Meier 5-year survival rates of the patients treated without chemotherapy and with chemotherapy were 50% and 76%, respectively. CONCLUSION: Limb salvage surgery with chemotherapy is a viable treatment option for patients with malignant fibrous histiocytoma of bone. It achieves higher survival rates than resection alone. Such therapy improves quality of life and provides a useful and functional limb.

The functional and oncological results after scapulectomy for scapular tumours: 2-16-year results.

Twenty-five patients underwent scapulectomy for various tumours between 1989 and 2005. We describe 23 patients with scapular tumours who were followed-up for a minimum of 2 years after scapulectomy. The average age was 29 years, and two-thirds of the patients were male. Nineteen patients had malignant neoplasms, of which chondrosarcoma was most common, followed by Ewing's sarcoma. Surgical staging was by Enneking's system, with stage IIB being the most frequent. Fifteen patients underwent total scapulectomy, and the rest had their glenoid retained. With an average follow-up period of 66.7 months (23-202 months), functional and oncological outcomes were evaluated for all patients. Two patients had superficial wound infections requiring antibiotics, and one had skin necrosis requiring skin cover. Functional outcome was satisfactory in 13 patients. Cosmetically and emotionally acceptable surgery, scapulectomy made 19 patients continuously disease free, while four patients died of disease. The 5-year survival rate of 19 patients with malignant tumours was 75.9%. Retention of the glenohumeral articulation (subtotal) resection gives superior functional results.

Paget's sarcoma: limb salvage by custom mega prosthesis: four case reports.

Of 4 Paget's sarcoma patients (age range, 55-68 years) underwent limb salvage surgery by custom mega prosthesis, 3 had lesions in the upper extremity and one in the proximal femur. Three of the patients were at stage IIB of the disease, according to Enneking's system of staging musculoskeletal tumours. All 4 patients underwent wide resection with a mean length of 152.5 mm. The defects were reconstructed with custom-made prostheses: proximal humeral prostheses in 2 of the patients, total elbow prosthesis in one, and total hip prosthesis in one. During a mean postoperative follow-up period of 40 months, one died of disseminated disease 14 months after surgery; one remained disease-free; 2 had local recurrence and required amputation, of whom one died of disseminated disease one year after amputation, the other had no further evidence of the disease. We report the functional outcomes of the 2 patients who were alive at the latest follow-up. The 2-year patient survival rate was 50%.

Proximal femoral reconstruction with custom mega prosthesis.

Seventy-seven patients with proximal femoral tumours underwent limb salvage surgery with custom mega prostheses between July 1989 and January 2002. We describe 44 cases with a mean follow-up of 57.8 months. Forty-one patients presented with malignant neoplasms of which 11 were malignant giant cell tumours, eight were chondrosarcoma and five were metastases. Wide margins were achieved for all malignant tumours, and marginal resection was done for all benign and metastatic lesions. Six patients died of disease and two were lost to follow-up. Mechanical complications were observed in six cases, fracture being the most common. Oncological complications occurred in five patients. Functional activity was satisfactory in 79% of patients.

The Saddle prosthesis in periacetabular tumours.

The Saddle endoprosthesis provides a means of establishing a stable and mobile articulation between the femur and a partially resected pelvis. Six patients with malignant or aggressive benign bone tumours underwent resection and replacement with custom-made Saddle endoprosthesis. Wide margin was achieved in four cases and marginal margin in two. Follow-up ranged from 24 to 41 months. All the six patients were then alive, five being disease-free. One patient developed deep infection and local recurrence necessitating removal of the prosthesis. The functional results were either excellent or good in five patients.

Lethality of a heat- and phosphate-catalyzed glucose by-product to Escherichia coli O157:H7 and partial protection conferred by the rpoS regulon.

A by-product of glucose produced during sterilization (121 degrees C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25 degrees C. An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25 degrees C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4 degrees C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E. coli O157:H7 and the protective effects afforded by rpoS-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media.

Gli genes and limb development.

During development of the limb Shh plays a key role as a mediator of zone of polarizing activity (ZPA). However, the molecular mechanisms by which Shh directs anterior/posterior patterning in the limb remain unknown. Members of the Gli gene family encode zinc-finger transcription factors and represent likely candidates for being regulators of Shh target genes. In this review we would like to summarize the current knowledge on expression and function of Gli genes in limb development.

Clinicopathologic study of lupus nephritis.

Needle biopsies of kidney were done in 35 cases of systemic lupus erythematosus (SLE) with renal lesions. The lupus nephritis were classified according to WHO classification and were correlated with response to therapy and prognosis. Detailed clinical features, routine haematological, biochemical tests (e.g., serum urea, creatinine, total protein and albumin, cholesterol, etc), examination of urine (degree of proteinuria and cells) and occurrence of various auto-antibodies e.g., antinuclear antibody (ANA), anti double stranded DNA (anti DsDNA) by enzyme immunoassay (EIA) method, LE cells and rheumatoid factor (RF) were studied in all cases. Clinically hypertension was present in 19 (54.3%) cases and nephrotic range of proteinuria was detected in 20 (57.2%) cases. ANA was found in 31 (88.5%) cases, anti DsDNA 24 (68.5%) and LE cells were detected in 25 (71.5%) cases. RF was detected in 2 (5.7%) cases. Histologically the most frequent lesions were class IV occurring in 15 cases (42.8%) with initial complete remission achieved only 4 cases by immunosuppressive therapy. Active lesions were also most frequent in this class. Class III lesions were found in 8 (22.8%) cases with 6 cases had complete remission. The best prognosis was noted in class II cases with 4 out of 5 (14.3%) cases had complete remission. Class V lesions were found in 6 (17.2%) cases with complete remission achieved in 3 cases. Only one patient presented with class VI lesion. RF positive cases had milder renal lesions.

Psychiatric disorder in essential dyspepsia.

We wanted to examine the prevalence of psychiatric morbidity in patients diagnosed as having essential dyspepsia, as well as the short-term course of dyspeptic symptoms, following drug treatment of the psychiatric condition. Seventy-four patients with essential dyspepsia presenting to the gastroenterology outpatient department of a medical college were investigated for the presence of psychiatric disorder. The response to an open trial of pharmacotherapy in 50 patients with a psychiatric disorder and no other demonstrable pathology was assessed. These patients met the criteria for a DSM-III-R diagnosis, most commonly major depressive disorder (26) or generalized anxiety disorder (10). The mean age of those with a psychiatric disorder alone was significantly higher than that of those with another demonstrable pathology. With treatment, 16 patients with no demonstrable pathology other than psychiatric disorder (depression: 12; anxiety: 4) showed improvement over a period of 6 weeks in psychiatric as well as dyspepsia ratings. The difference was however statistically significant only for the group with major depressive disorder. We concluded that, despite differences in the characteristics of the population studied, a psychiatric diagnosis is associated with at least a proportion of cases with essential dyspepsia and emerges as a likely explanation.

Characteristics of unmarried adolescent mothers: determinants of child rearing versus adoption.

The sociodemographic and biographical characteristics of a group of unmarried adolescent mothers who opted to rear their children were compared with those of a matched group who placed their children for adoption. Those choosing adoption were more likely to be of higher socioeconomic status, to have higher educational aspirations, and to be from suburban residences. Suggestions for intervention and appropriate services are offered.

Bishnupur achromats and their relatives (an exploratory study with six colour vision tests).

Thirteen subjects from the 'Sankhabaniks' of Bishnupur and two new similar cases were given six colour vision tests. All had photophobia, fixation nystagmus, low visual acuity and marked, though not complete, loss of colour sense. Forty other males and 24 females related to the defectives were also tested with at least five of the tests, for comparison. The tests were Ishihara, HRR test, Sloan's Achromatopsia test, the Dichotomous (D 15) test, Hundred Hue test and the Pickford-Nicolson Anomaloscope. The present research confirmed the provisional conclusion of Bose et al. (1968) that the achromatopsia in Bishnupur is an autosomal recessive character. That women relatives of the achromats showed greater average error scores with the Dichotomous test, the Hundred Hue test and the Sloan's test than male relatives, suggests that the defect is more readily manifested in males, and that the female relatives would include a number of genetic defectives with incomplete manifestation due to sex control. The defectives were clearly distinguished from the relatives as a group.

Colour vision tests and colour naming by thirteen incomplete achromats in Bishnupur.

As an exploratory study six colour vision tests were given to nine male and two female achromats from the Shankhabanik community in Bishnupur, and to two additional similar males. All thirteen subjects had severe photophobia, fixation nystagmus, extreme weakness of vision (4/24 to 3/60) and the red end of the spectrum was much shortened. This research indicates that they had a form of incomplete achromatopsia, varying from an almost complete to a very severe partial loss of colour vision. The condition is inherited as an autosomal recessive. The most likely interpretation of these cases is that they are incomplete rod achromats. Their performance on the colour vision tests is tabulated, and shows complete inability to do the Ishihara test; nearly complete inability on the HRR test, with a possible slight tendency to do better in the yellow-blue than the red-green sub-tests; on Sloan's test they show approximate accordance with her results for achromats; they have severe difficulty with the dichotomous and 100-hue tests, with a possible slight tendency to make fewer errors on the G/B sections. The anomaloscope shows little abnormality of mid-matching points, but great increases in average matching ranges above the normal, although not absolute loss of colour sense, but with extreme darkening or shortening of the red end of the spectrum. Their colour naming was carefully recorded, and was fairly good occasionally, sometimes erroneous without being wildly at fault, and most often completely wrong. The records of colour naming were made, not, of course, as a form of colour vision test, but simply to illustrate the ways in which such defectives make an effort to use colour names in general use among their friends and relatives.

Incomplete achromatopsia in Bishnupur.

Nine males and 2 females from the Shankhabanik Community in Bishnupur, provisionally diagnosed as incomplete rod achromats by Bose, Joardar and Sukul in 1968, with 2 new similar males, were tested more fully with six colour vision tests. All had photophobia, nystagmus of fixation, extremely low visual acuity and extreme loss of colour sense with shortened red spectrum. 40 other males and 24 females, relatives of the defectives, were also tested for comparison. The provisional diagnosis was confirmed, and the hypothesis of autosomal inheritance seemed most probably true. Various details about the relatives emerged.