Long-term Results of Splenectomy in Transfusion-dependent Thalassemia.

Splenectomy is indicated in transfusion-dependent thalassemia (TDT) only in certain situations. This study aimed to present the effectiveness, complications, and long-term follow-up results of splenectomy in children with TDT. We performed a 30-year single-institution analysis of cases of splenectomy for TDT between 1987 and 2017 and their follow-up until 2021. A total of 39 children (female/male: 24/15) were included. The mean age at splenectomy was 11.2±3.2 years, and their mean follow-up duration after splenectomy was 21.5±6.4 years. Response was defined according to the patient's annual transfusion requirement in the first year postsplenectomy and on the last follow-up year. Complete response was not seen in any of the cases; partial response was observed in 32.3% and no response in 67.6%. Thrombocytosis was seen in 87% of the patients. The platelet counts of 7 (17.9%) patients were >1000 (10 9 /L), and aspirin prophylaxis was given to 22 (56.4%) patients. Complications were thrombosis in 2 (5.1%) patients, infections in 11 (28.2%) patients, and pulmonary hypertension in 4 (10.2%) patients. Our study showed that after splenectomy, the need for transfusion only partially decreased in a small number of TDT patients. We think splenectomy can be delayed with appropriate chelation therapy up to higher annual transfusion requirement values.

Clinical Features and Outcome of Children With Hereditary Spherocytosis.

OBJECTIVE: The aim of this study was to evaluate the demographics, clinical, and laboratory findings and treatment responses of patients with hereditary spherocytosis (HS). MATERIALS AND METHODS: Data of children with HS were examined. Diagnosis was based on clinical history, physical examination, family history, presence of spherocytes on peripheral blood smear, and osmotic fragility test. RESULTS: A total of 101 patients were included. The median (range) age at diagnosis was 38.0 (1 to 188) months. Mild, moderate, and severe forms of HS were present in 29 (28.7%), 15 (14.9%), and 57 (56.4%) patients, respectively. Family history was available in 73 patients and 56 of these (76.7%) had a positive family history for HS. Forty-five (44.5%) patients needed regular transfusions and all of these had severe disease. Although most patients did not require transfusion postsplenectomy, 2 of 45 (4.4%) patients continued to require transfusion. Transfusion dependence was significantly (P<0.001) higher in patients with severe spherocytosis. CONCLUSIONS: In HS, splenomegaly, pallor, and jaundice are the most common clinical features. Splenectomy dramatically reduces hemolysis in most cases and virtually abolishes further requirement for transfusion.

Hepatitis-Associated Aplastic Anemia: Etiology, Clinical Characteristics and Outcome.

Hepatitis-associated aplastic anemia (HAA) is a form of acquired aplastic anemia (AA) in which bone marrow failure develops after an acute attack of hepatitis. Bone marrow failure leading to AA is generally severe in cases of HAA and fatal if left untreated. This retrospective multicenter study investigated clinical and laboratory characteristics, possible causes, treatment, and outcome of HAA in children. Twenty patients from 8 centers were included in the study. Aspartate aminotransferase and alanine aminotransferase were <3 to 5×upper limit of normal (ULN) in 2 patients, <5 to 10×ULN in 2 patients, and >10×ULN in 16 patients. Acute liver failure developed in 5 (29%) patients. Pancytopenia was simultaneously present in 6 of 20 (30%) patients. Eleven of the 20 patients (55%) were alive, in remission and transfusion free. Those who were alive either had undergone hematopoietic stem cell transplantation and/or immunosuppressive treatment, except 1 patient who had received no treatment. Patients with the diagnosis of acute hepatitis should be evaluated and followed up carefully for presence of cytopenia, so that definitive treatment of AA can be initiated in a timely and appropriate manner when needed.

Doppler assessment of the fetus in pregnant women recovered from COVID-19.

OBJECTIVE: The aim of this study was to evaluate the maternal-fetal Doppler patterns in pregnant women recovered from COVID-19. METHODS: This prospective case-control study was conducted in Ankara City Hospital between July 1, 2020 and August 30, 2020. Thirty pregnant women who were diagnosed with COVID-19 and completed the quarantine process were compared with 40 healthy pregnant women in terms of the fetal Doppler parameters. All pregnant women diagnosed with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were followed up in our clinic and their diagnoses have been confirmed in nasopharyngeal and oropharyngeal samples by quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) method. Doppler ultrasonographic assessment of the uterine arteries (UtA) and middle cerebral artery (MCA) were used in addition to umbilical artery (UA) Doppler between 23 and 40 weeks of gestation. Also, cerebroplacental ratio (CPR) was calculated according to gestational age. RESULTS: The pulsatility and resistance indices of umbilical and UtA showed a significant increase in pregnant women in the study group compared to the control group (p < 0.05). Multivariable logistic regression analysis revealed that pulsatility and resistance indices of the mean UtA were independently associated with disease (OR > 1000, 95%CI 9.77 to >1000, p = 0.009; OR 0,000 95%CI 0,000-0,944, p = 0,049), respectively. Medical treatment was given to 16/30 (53%) of pregnant women diagnosed with COVID-19. CONCLUSION: In conclusion, uterine artery Doppler indices in the third trimester may have clinical value in pregnant women recovered from COVID-19.

The Efficacy of Oral Vitamin B12 Replacement for Nutritional Vitamin B12 Deficiency.

Standard treatment of vitamin B12 deficiency has not been well established in childhood, the ideal amount of supplemental vitamin B12 is not clear. Vitamin B12 deficiency is classically treated with intramuscular injections. In this study, we aimed to investigate the efficacy of oral therapy in children with vitamin B12 deficiency. Patients with serum cobalamin concentrations <300 pg/mL aged between 6 months to 18 years were included in this prospective study. Children were treated orally either with a combination of multivitamin tablet daily or vitamin B12 ampules. Serum specimens were obtained at the end of first and third months of treatment for vitamin B12 levels. A total of 79 patients were included in the study. The mean pretreatment vitamin B12 level increased from 182±47.6 pg/mL to 482±318 pg/mL after 1 month of treatment in the whole cohort. Comparison of the pretreatment vitamin B12 levels with first and third month posttreatment values showed significant difference (P-value, 0.001 and 0.028, respectively). In this study, oral cyanocobalamin was found effective for the treatment of vitamin B12 deficiency in children.

Regulation of high glucose-induced apoptosis of brain pericytes by mitochondrial CA VA: A specific target for prevention of diabetic cerebrovascular pathology.

Events responsible for cerebrovascular disease in diabetes are not fully understood. Pericyte loss is an early event that leads to endothelial cell death, microaneurysms, and cognitive impairment. A biochemical mechanism underlying pericyte loss is rapid respiration (oxidative metabolism of glucose). This escalation in respiration results from free influx of glucose into insulin-insensitive tissues in the face of high glucose levels in the blood. Rapid respiration generates superoxide, the precursor to all reactive oxygen species (ROS), and results in pericyte death. Respiration is regulated by carbonic anhydrases (CAs) VA and VB, the two isozymes expressed in mitochondria, and their pharmacologic inhibition with topiramate reduces respiration, ROS, and pericyte death. Topiramate inhibits both isozymes; therefore, in the earlier studies, their individual roles were not discerned. In a recent genetic study, we showed that mitochondrial CA VA plays a significant role in regulation of reactive oxygen species and pericyte death. The role of CA VB was not addressed. In this report, genetic knockdown and overexpression studies confirm that mitochondrial CA VA regulates respiration in pericytes, whereas mitochondrial CA VB does not contribute significantly. Identification of mitochondrial CA VA as a sole regulator of respiration provides a specific target to develop new drugs with fewer side effects that may be better tolerated and can protect the brain from diabetic injury. Since similar events occur in the capillary beds of other insulin-insensitive tissues such as the eye and kidney, these drugs may also slow the onset and progression of diabetic disease in these tissues.

Utility of baseline serum phosphorus levels for predicting remission in acromegaly patients.

PURPOSE: High GH and IGF I levels increase tubular phosphate reabsorption in patients with acromegaly. We aimed to investigate the utility of serum phosphorus levels as an indicator for predicting chance of remission in acromegaly patients. DESIGN: Fifty-one patients (n: 51; F: 24, M: 27) with diagnosis of acromegaly were included in the study. Plasma IGF-1, Phosphorus (P) and nadir GH levels on oral glucose tolerance test (OGTT) at the time of diagnosis were analysed retrospectively. Patients were classified into two groups according to their plasma P levels; P ≤ 4.5 mg/dl (Group-1, n: 23, 45.1%), P > 4.5 mg/dl (Group-2, n: 28, 54.9%). Two groups were compared according to remission status; remission (n: 27) and non-remission (n: 24). Remission was defined with absence of clinical symptoms, normal plasma IGF-1 (adjusted for age and gender) and GH levels (<1 mcg/dl) at least 3 months after initial treatment. RESULTS: Serum P levels decreased significantly after treatment in both groups (p < 0.001). There was a significant correlation between baseline phosphorus levels and remission rates, nadir GH in OGTT, pituitary adenoma size and Ki-67 scores (p = 0.001, r: -0.51; p = 0.01, r: 0.44; p = 0.001, r: 0.52; p = 0.02, r: 0.71, respectively). Mean baseline P levels were significantly higher in patients with non-remission (4.8 vs 4.2, P < 0.001). Logistic regression analysis did not reveal an independent effect on remission with any of these risk factors. CONCLUSION: High serum P levels may be an indicator for a low likelihood of onset of remission in acromegaly patients. Further studies with wider spectrum are needed to make specific suggestions.

INCIDENTAL PAGET'S DISEASE DISGUISED AS BONE METASTASIS IN A PATIENT WITH ENDOMETRIUM CARCINOMA.

Paget's disease is a disorder of aging bone which occurs in the setting of accelarated bone remodelling. In the presented case we discuss the difficulties in the diagnosis of Paget's disease in a 77 year old patient with coexisting endometrium carcinoma. The patient was initially diagnosed with metastatic bone disease due to endometrium adenocarcinoma when she was admitted to oncology clinic with pelvic pain. Bone scintigraphy with Tc99 and (18)F fluorodeoxyglucose positron emission tomography/CT revealed an increased uptake on the bone lesions which were reported as metastatic bone involvement. Although the (18)F-FDG uptake was much higher than the levels that would generally be anticipated in a case with Paget's disease, high levels of bone turnover markers indicated further evaluation in the differential diagnosis and the definitive diagnosis of Paget's disease was established with the pathological evaluation of bone biopsy.

Blood-Brain Barrier Disruption and Neurovascular Unit Dysfunction in Diabetic Mice: Protection with the Mitochondrial Carbonic Anhydrase Inhibitor Topiramate.

All forms of diabetes mellitus are characterized by chronic hyperglycemia, resulting in the development of a number of microvascular and macrovascular pathologies. Diabetes is also associated with changes in brain microvasculature, leading to dysfunction and ultimately disruption of the blood-brain barrier (BBB). These changes are correlated with a decline in cognitive function. In diabetes, BBB damage is associated with increased oxidative stress and reactive oxygen species. This occurs because of the increased oxidative metabolism of glucose caused by hyperglycemia. Decreasing the production of bicarbonate with the use of a mitochondrial carbonic anhydrase inhibitor (mCAi) limits oxidative metabolism and the production of reactive oxygen species. In this study, we have demonstrated that 1) streptozotocin-induced diabetes resulted in BBB disruption, 2) ultrastructural studies showed a breakdown of the BBB and changes to the neurovascular unit (NVU), including a loss of brain pericytes and retraction of astrocytes, the two cell types that maintain the BBB, and 3) treatment with topiramate, a mCAi, attenuated the effects of diabetes on BBB disruption and ultrastructural changes in the neurovascular unit.

Targeted mutagenesis of mitochondrial carbonic anhydrases VA and VB implicates both enzymes in ammonia detoxification and glucose metabolism.

Prior studies with carbonic anhydrase (CA) inhibitors implicated mitochondrial CA in ureagenesis and gluconeogenesis. Subsequent studies identified two mitochondrial CAs. To distinguish the contribution of each enzyme, we studied the effects of targeted disruption of the murine CA genes, called Car5A and Car5B. The Car5A mutation had several deleterious consequences. Car5A null mice were smaller than wild-type littermates and bred poorly. However, on sodium-potassium citrate-supplemented water, they produced offspring in expected numbers. Their blood ammonia concentrations were markedly elevated, but their fasting blood sugars were normal. By contrast, Car5B null mice showed normal growth and normal blood ammonia levels. They too had normal fasting blood sugars. Car5A/B double-knockout (DKO) mice showed additional abnormalities. Impaired growth was more severe than for Car5A null mice. Hyperammonemia was even greater as well. Although fertile, DKO animals were produced in less-than-predicted numbers even when supplemented with sodium-potassium citrate in their drinking water. Survival after weaning was also reduced, especially for males. In addition, fasting blood glucose levels for DKO mice were significantly lower than for controls (153 ± 33 vs. 230 ± 24 mg/dL). The enhanced hyperammonemia and lower fasting blood sugar, which are both seen in the DKO mice, indicate that both Car5A and Car5B contribute to both ammonia detoxification (ureagenesis) and regulation of fasting blood sugar (gluconeogenesis). Car5A, which is expressed mainly in liver, clearly has the predominant role in ammonia detoxification. The contribution of Car5B to ureagenesis and gluconeogenesis was evident only on a Car5A null background.

Impact of a community-based perinatal and newborn preventive care package on perinatal and neonatal mortality in a remote mountainous district in Northern Pakistan.

BACKGROUND: There is limited evidence from community-based interventions to guide the development of effective maternal, perinatal and newborn care practices and services in developing countries. We evaluated the impact of a low-cost package of community-based interventions implemented through government sector lady health workers (LHWs) and community health workers (CHWs) of a NGO namely Aga Khan Health Services on perinatal and neonatal outcomes in a sub-population of the remote mountainous district of Gilgit, Northern Pakistan. METHODS: The package was evaluated using quasi experimental design included promotion of antenatal care, adequate nutrition, skilled delivery and healthy newborn care practices. Control areas continued to receive the routine standard health services. The intervention areas received intervention package in addition to the routine standard health services. Outcome measures included changes in maternal and newborn-care practices and perinatal and neonatal mortality rates between the intervention and control areas. RESULTS: The intervention was implemented in a population of 283324 over a 18 months period. 3200 pregnant women received the intervention. Significant improvements in antenatal care (92% vs 76%, p < .001), TT vaccination (67% vs 47%, p < .001), institutional delivery (85% vs 71%, p < .001), cord application (51% vs 71%, p < .001), delayed bathing (15% vs 43%, p < .001), colostrum administration (83% vs 64%, p < .001), and initiation of breastfeeding within 1 hour after birth (55% vs 40%, p < .001) were seen in intervention areas compared with control areas. Our results indicate significant reductions in mortality rates in intervention areas as compared to control areas from baseline in perinatal mortality rate (from 47.1 to 35.3 per 1000 births, OR 0.62; 95% CI: 0.56-0.69; P 0.02) and neonatal mortality rates (from 26.0 to 22.8 per 1000 live births, 0.58; 95% CI: 0.48-0.68; P 0.03). CONCLUSIONS: The implementation of a set of low cost community-based intervention package within the health system settings in a mountainous region of Pakistan was found to be both feasible and beneficial. The interventions had a significant impact in reduction of the burden of perinatal and neonatal mortality. TRIAL REGISTRATION: This study is registered, ClinicalTrial.gov NCT02412293 .

Identification of ATP8B1 as a blood-brain barrier-enriched protein.

In order to define the molecular anatomy of the blood-brain barrier (BBB) that may be relevant to either barrier or transport function, proteins that are overexpressed in the cerebral microvessels should be identified. We used differential display to identify novel proteins that are overexpressed or unique to the BBB. DNA sequence analysis is one of the differentially expressed transcripts showed that it is highly homologous with the ATPase class I, type 8B, and member 1 (ATP8B1) protein and contains an ATPase domain and a phospholipid-binding domain. ATP8B1 is expressed in the BBB microvessels but not brain tissue lacking microvessels. Likewise, ATP8B1 was enriched in BBB microvessels similar to glucose transporter 1. Immunohistochemistry using an ATP8B1-specific antibody demonstrated preferential staining of the microvessels within the cerebral tissue. These results suggest that ATP8B1, a P-type aminophospholipid translocase, is enriched in cerebral microvessels and may have a role in plasma membrane lipid transport.

NMDA receptor-dependent afterdepolarizations are curtailed by carbonic anhydrase 14: regulation of a short-term postsynaptic potentiation.

In the hippocampus, extracellular carbonic anhydrase (Car) speeds the buffering of an activity-generated rise in extracellular pH that impacts H(+)-sensitive NMDA receptors (NMDARs). We studied the role of Car14 in this brain structure, in which it is expressed solely on neurons. Current-clamp responses were recorded from CA1 pyramidal neurons in wild-type (WT) versus Car14 knock-out (KO) mice 2 s before (control) and after (test) a 10 pulse, 100 Hz afferent train. In both WT and KO, the half-width (HW) of the test response, and its number of spikes, were augmented relative to the control. An increase in presynaptic release was not involved, because AMPAR-mediated EPSCs were depressed after a train. The increases in HW and spike number were both greater in the Car14 KO. In 0 Mg(2+) saline with picrotoxin (using a 20 Hz train), the HW measures were still greater in the KO. The Car inhibitor benzolamide (BZ) enhanced the test response HW in the WT but had no effect on the already-prolonged HW in the KO. With intracellular MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]-cyclohepten-5,10-imine maleate], the curtailed WT and KO responses were indistinguishable, and BZ caused no change. In contrast, the extracellular alkaline changes evoked by the train were not different between WT and KO, and BZ amplified these alkalinizations similarly. These data suggest that Car14 regulates pH transients in the perisynaptic microenvironment and govern their impact on NMDARs but plays little role in buffering pH shifts in the broader, macroscopic, extracellular space.

A dose-finding study of temsirolimus and liposomal doxorubicin for patients with recurrent and refractory bone and soft tissue sarcoma.

There are few effective therapies for high-risk sarcomas. Initial chemosensitivity is often followed by relapse. In vitro, mammalian target of rapamycin (mTOR) inhibition potentiates the efficacy of chemotherapy on resistant sarcoma cells. Although sarcoma trials using mTOR inhibitors have been disappointing, these drugs were used as maintenance. We conducted a Phase I/II clinical trial to test the ability of temsirolimus to potentiate the cytotoxic effect of liposomal doxorubicin and present here the dose-finding portion of this study. Adult and pediatric patients with recurrent or refractory sarcomas were treated with increasing doses of liposomal doxorubicin and temsirolimus using a continual reassessment method for escalation, targeting a dose-limiting toxicity rate of 20%. Blood samples were drawn before and after the first dose of temsirolimus in Cycles 1 and 2 for pharmacokinetic analysis. The maximally tolerated dose combination was liposomal doxorubicin 30 mg/m(2) monthly with temsirolimus 20 mg/m(2) weekly. Hematologic toxicity was common but manageable. Dose-limiting toxicities were primarily renal. Concurrent administration of liposomal doxorubicin resulted in increased exposure to sirolimus, the active metabolite of temsirolimus. Thus, the combination of liposomal doxorubicin and temsirolimus is safe for heavily pretreated sarcoma patients. Co-administration with liposomal doxorubicin did not alter temsirolimus pharmacokinetics, but increased exposure to its active metabolite.

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes.

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood-brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.

Pharmacological inhibition of mitochondrial carbonic anhydrases protects mouse cerebral pericytes from high glucose-induced oxidative stress and apoptosis.

Diabetes-associated complications in the microvasculature of the brain are caused by oxidative stress, generated by overproduction of reactive oxygen species from hyperglycemia-induced accelerated oxidative metabolism of glucose. Pericytes, essential for the viability of the microvasculature, are especially susceptible to oxidative stress. Mitochondrial carbonic anhydrases, regulators of the oxidative metabolism of glucose, determine the rate of reactive oxygen species production and inhibition of mitochondrial carbonic anhydrases rescues glucose-induced pericyte loss in the diabetic mouse brain. We hypothesized that high glucose induces intracellular oxidative stress and pericyte apoptosis and that inhibition of mitochondrial carbonic anhydrases protects pericytes from oxidative stress-induced apoptosis. To validate our hypothesis, conditionally immortalized cerebral pericyte (IPC) cultures were established from Immortomice to investigate the effect of high glucose on oxidative stress and pericyte apoptosis. The IPCs expressed pericyte markers and induced high transendothelial electrical resistance and low permeability in brain endothelial cell monolayers comparable with pericytes in primary cultures. The IPCs also secreted cytokines constitutively and in response to lipopolysaccharide similar to pericytes. High glucose caused oxidative stress and apoptosis of these cells, with both oxidative stress and apoptosis significantly reduced after mitochondrial carbonic anhydrase inhibition. These results provide the first evidence that pharmacological inhibition of mitochondrial carbonic anhydrases attenuates pericyte apoptosis caused by high glucose-induced oxidative stress. Carbonic anhydrase inhibitors have a long history of safe clinical use and can be immediately evaluated for this new indication in translational research. Thus, mitochondrial carbonic anhydrases may provide a new therapeutic target for oxidative stress-related illnesses of the brain.

Progressive renal injury from transgenic expression of human carbonic anhydrase IV folding mutants is enhanced by deficiency of p58IPK.

Mutations in the human carbonic anhydrase IV (hCAIV) have been associated with retinal degeneration in an autosomal-dominant form of retinitis pigmentosa (RP17). Prior in vitro cell culture studies confirmed that all of the RP17-associated hCAIV mutations cause protein misfolding, leading to endoplasmic reticulum (ER) stress-induced apoptosis in cells expressing the mutant proteins. To evaluate the physiological impacts of these folding mutants in other carbonic anhydrase IV-producing tissues, we generated two transgenic mouse lines expressing R219S or R14W hCAIV under control of the endogenous hCAIV promoter. Expression of either of these mutant proteins in kidneys caused progressive renal injury in male transgenic mice as evidenced by an age-dependent increase in the tubule cell apoptosis starting at approximately 20 weeks of age and vacuolization throughout the renal cortex in older mice. Up-regulation of the ER chaperone, BiP, was observed in the cells of the renal cortex of the male transgenic mice, suggesting ER stress as a causal factor for the renal injury. The renal injury inflicted by expression of the folding mutants was markedly enhanced by haploinsufficiency of the ER cochaperone p58(IPK). The transgenic mice expressing the hCAIV folding mutants on a p58(IPK) heterozygous background showed extensive renal tubular apoptosis by approximately 10 weeks of age in both male and female mice. These data indicate that expression of the RP17-associated folding mutants of hCAIV can adversely affect tissues beyond the retina and their in vivo proteotoxicity is sensitive to modulation of the protein folding environment of the ER.

Pathogenesis of retinitis pigmentosa associated with apoptosis-inducing mutations in carbonic anhydrase IV.

Missense mutations in the carbonic anhydrase IV (CA IV) gene have been identified in patients with an autosomal dominant form of retinitis pigmentosa (RP17). We used two transient expression systems to investigate the molecular mechanism by which the newly identified CA IV mutations, R69H and R219S, contribute to retinal pathogenesis. Although the R219S mutation drastically reduced the activity of the enzyme, the R69H mutation had a minimal effect, suggesting that loss of CA activity is not the molecular basis for their pathogenesis. Defective processing was apparent for both mutant proteins. Cell surface-labeling techniques showed that the R69H and R219S mutations both impaired the trafficking of CA IV to the cell surface, resulting in their abnormal intracellular retention. Expression of both CA IV mutants induced elevated levels of the endoplasmic reticulum (ER) stress markers, BiP and CHOP, and led to cell death by apoptosis. They also had a dominant-negative effect on the secretory function of the ER. These properties are similar to those of R14W CA IV, the signal sequence variant found in the original patients with RP17. These findings suggest that toxic gain of function involving ER stress-induced apoptosis is the common mechanism for pathogenesis of this autosomal-dominant disease. Apoptosis induced by the CA IV mutants could be prevented, at least partially, by treating the cells with dorzolamide, a CA inhibitor. Thus, the use of a CA inhibitor as a chemical chaperone to reduce ER stress may delay or prevent the onset of blindness in RP17.

Assessments of some trace metals in water samples of nursery pond of Grass Carp (Ctenopharyngodon idella, Valenciennes, 1844) in Bannu Fish Hatchery of Khyber Pakhtunkhwa, Pakistan.

The present investigation was aimed to examine the concentrations of trace metals including e.g copper (Cu), manganese (Mn), nickel (Ni), and zinc (Zn) in water samples collected from nursery pond of grass carp (Ctenopharyngodon idella) in Bannu Hatchery of Khyber Pakhtunkhwa during the period from April 2018 to January 2019. The temperature and pH of each water sample were measured for the whole study duration. The concentration of Copper (Cu), Manganese (Mn), Nickel (Ni), and Zinc (Zn) in collected water samples were measured in mg/liter by using Atomic Absorption Spectrophotometer. The blank and standard solutions for device calibration Standard solutions i.e., 2.0 mg, 4.0 mg, and 6.0 were used to measure the concentration of these metals in water samples to verify the measurements. The data was statistically analyzed on descriptive statistics (estimation of proportions and standard deviation) used to summarize mean concentration. The results obtained of both temperature and pH of water samples were found in ranged 10 to 36 0C and 7.0 to 8.44; whereas the size of fry stages was ranged from 4.0 to 56.0 mm in total length. The results of investigated metals found in pond water samples are in order of Zn>Mn>Ni>Cu, respectively. As optimum temperature and pH for grass carp were mostly between 15 0C and 30 0C and pH 6.5 to 8.0. It was concluded from obtained results that temperature, pH, and trace metals were found appropriate for the growth of Ctenopharyngodon idella from fry to fingerling stages, but the highest amount of zinc can cause its mortality. It is a preliminary study on grass carp culturing in Bannu fish hatchery so, it would provide useful information for model fish seed production unit in a hatchery.

Infused Fc-tagged beta-glucuronidase crosses the placenta and produces clearance of storage in utero in mucopolysaccharidosis VII mice.

Glycosaminoglycan storage begins in prenatal life in patients with mucopolysaccharidosis (MPS). In fact, prenatal hydrops is a common manifestation of MPS VII because of beta-glucuronidase (GUS) deficiency. One way to address prenatal storage might be to deliver the missing enzyme across the placenta into the fetal circulation. Maternal IgG is transported across the placenta by the neonatal Fc receptor (FcRn), which recognizes the Fc domain of IgG and mediates transcytosis from maternal to fetal circulation. We hypothesized that we could exploit this process to deliver corrective enzyme to the fetus. To test this hypothesis, the C-terminal fusion protein, GUS-Fc, was compared with native, untagged, recombinant GUS for clearance from the maternal circulation, delivery to the fetus, and reduction of lysosomal storage in offspring of MPS VII mice. We observed that GUS-Fc, infused into pregnant mothers on embryonic days 17 and 18, was transported across the placenta. Similarly infused untagged GUS was not delivered to the fetus. GUS-Fc plasma enzyme activity in newborn MPS VII mice was 1,000 times that seen after administration of untagged GUS and approximately 100 times that of untreated WT newborns. Reduced lysosomal storage in heart valves, liver, and spleen provided evidence that in utero enzyme replacement therapy with GUS-Fc targeted sites of storage in the MPS VII fetus. We hypothesize that this noninvasive approach could deliver the missing lysosomal enzyme to a fetus with any lysosomal storage disease. It might also provide a method for inducing immune tolerance to the missing enzyme or another foreign protein.