Endosomal Acid-Base Homeostasis in Neurodegenerative Diseases.

Neurodegenerative disorders are debilitating and largely untreatable conditions that pose a significant burden to affected individuals and caregivers. Overwhelming evidence supports a crucial preclinical role for endosomal dysfunction as an upstream pathogenic hub and driver in Alzheimer's disease (AD) and related neurodegenerative disorders. We present recent advances on the role of endosomal acid-base homeostasis in neurodegeneration and discuss evidence for converging mechanisms. The strongest genetic risk factor in sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4), which potentiates pre-symptomatic endosomal dysfunction and prominent amyloid beta (Aß) pathology, although how these pathways are linked mechanistically has remained unclear. There is emerging evidence that the Christianson syndrome protein NHE6 is a prominent ApoE4 effector linking endosomal function to Aß pathologies. By functioning as a dominant leak pathway for protons, the Na+/H+ exchanger activity of NHE6 limits endosomal acidification and regulates ß-secretase (BACE)-mediated Aß production and LRP1 receptor-mediated Aß clearance. Pathological endosomal acidification may impact both Aß generation and clearance mechanisms and emerges as a promising therapeutic target in AD. We also offer our perspective on the complex role of endosomal acid-base homeostasis in the pathogenesis of neurodegeneration and its therapeutic implications for neuronal rescue and repair strategies.

Differential Gene Expression Analysis of Human Ovarian Follicular Cumulus and Mural Granulosa Cells Under the Influence of Insulin in IVF Ovulatory Women and Polycystic Ovary Syndrome Patients Through Network Analysis.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a commonly occurring reproductive disorder among the reproductive-aged women. Its global occurrence varies based on diagnostic guidelines, ethnicities, and locations of concern. Insulin resistance (IR) is commonly observed around 65-70% of women diagnosed with PCOS, representing a prevalent association. Consequently, the study was designed with an objective of illustrating the effect of insulin on mural and cumulus granulosa cells (GCs) of PCOS patients in comparison to normal ovulating women. METHODOLOGY: This study is a case-control design, wherein a total of 80 participants were recruited meeting criterion of inclusion and exclusion, divided into 8 groups with each group consisting of 10 samples. The process involves the isolation and culturing of mural granulosa cells (MGC) and cumulus granulosa cells (CGC) with and without exposure to insulin. The proteins released by untreated GCs and insulin-treated GCs were extracted, and complex protein mixtures were digested with trypsin, followed by tandem mass spectrometry analysis and data processing using bioinformatics. RESULTS: We found 595 proteins in both control and PCOS samples, of which 310 were contributed by MGCs and 285 by CGCs. The PCOS MGCs expressed 20%, both the normal MGCs and CGCs have equal representation of 16% by each, whereas the PCOS CGCs proteins contributed 15% of the total of the proteomic expression. However, the poor expression observed with the Insulin exposure, the Insulin treated PCOS CGCs contributes 13%, PCOS MGCs contributes 8%. The normal MGCs upon the Insulin treatment give 8% then and there only 4% of proteins expressed by normal CGCs after Insulin treatment. The Venn analysis widened on their precise expression topographies. The examination of strings exhibited important protein-protein interaction pathways. CONCLUSION: This is a pioneering investigation aimed to establish the link between hyperinsulinemia in localized follicular GCs and PCOS mechanisms by comparing them to control group. The examination of various attributes, mechanisms, and traits shown by genes and proteins in individuals with PCOS compared to control populations, alongside the investigation of the dynamics of these genes and proteins following exposure to insulin, holds promise for the formulation of novel hypotheses and strategies in the identification of new biomarkers.

Prospective observational study on clinical and epidemiological profile of adult patients presenting to the emergency department with suspected upper gastrointestinal bleed.

BACKGROUND AND OBJECTIVE: Bleeding from the upper gastrointestinal (GI) tract is one of the common medical emergencies. In this study, we assessed patients' socio-demographic and clinical characteristics and the association of clinical characteristics with treatment outcomes among patients with suspected upper gastrointestinal bleed (UGIB) presenting to the emergency department (ED). At present, there is a scarcity of data on UGIB in Northern part of India. MATERIAL AND METHOD: The study was a single-center, prospective observational study conducted at an urban tertiary care center. Consecutive patients with suspected UGIB were enrolled in the study from August 2020 to February 2022. A detailed history was obtained, including demographic data such as age and sex, presenting complaints, history of presenting illness, history related to co-morbidities, addiction, and drug history. Pre-endoscopic Rockall and Glasgow-Blatchford Score were calculated for each patient. The patients were subsequently followed up till discharge from the hospital. The final outcomes with regard to mortality, need for blood transfusion, length of emergency department stay, and discharge were noted. RESULT: 141 patients were included in the study. The mean age of the patients with suspected UGIB was 48 ± 14 years. 115 (81.6%) patients were male. The most common co-morbidity was chronic liver disease (40;28.4%). The most frequent presenting complaint in this study was hematemesis (96; 68.1%), followed by melena (76;53.9%). The mean (Standard Deviation, SD) of the Rockall Score was 2.46 ± 1.75. The mean (SD) of the Glasgow Blatchford Score was 12.46 ± 3.15 in patients with UGIB. CONCLUSION: In our study, hematemesis was the most prevalent symptom of suspected UGIB, followed by melena. Portal hypertension was the most common cause of UGIB. Most frequent comorbidities in patients suspected of UGIB were alcohol intake, Nonsteriodal Antiinflammatory Drugs (NSAIDs) abuse, and co-morbidities such as underlying chronic liver disease, hypertension, and diabetes. Early endoscopy can be of great utility to reduce morbidity and mortality.

Stereospecific Conversion of Boronic Esters into Enones using Methoxyallene: Application in the Total Synthesis of 10-Deoxymethynolide.

Enones are widely utilized linchpin functional groups in chemical synthesis and molecular biology. We herein report the direct conversion of boronic esters into enones using commercially available methoxyallene as a three-carbon building block. Following boronate complex formation by reaction of the boronic ester with lithiated-methoxyallene, protonation triggers a stereospecific 1,2-migration before oxidation generates the enone. The protocol shows broad substrate scope and complete enantiospecificity is observed with chiral migrating groups. In addition, various electrophiles could be used to induce 1,2-migration and give a much broader range of α-functionalized enones. Finally, the methodology was applied to a 14-step synthesis of the enone-containing polyketide 10-deoxymethynolide.

The Dowd-Beckwith Reaction: History, Strategies, and Synthetic Potential.

Ring-expansion strategies are valuable synthetic tools that take benefit of existing ring structures and evade the unfavorable enthalpic-and entropic effects that arise with end-to-end cyclizations. One potentially important class of such reactions is the Dowd-Beckwith reaction, the ring-expansion of ketones via alkoxy radicals. The exciting advancement in this research area is starting to show its potential, as demonstrated by applying this methodology in strategy-level bond formation to synthesize complex natural products. This Review aims to provide the first comprehensive survey of the development of the Dowd-Beckwith reaction spanning three decades from the initial report to the present day, thus providing the readers with great detail about the contributions of this reaction to organic synthesis. We hope that this review will further disclose the salient features of the Dowd-Beckwith reaction for synthetic applications and encourage the development of new, more advanced applications.

Twelve-month evaluation of a novel mineral-organic adhesive material used to stabilize dental implants placed in oversized osteotomies in vivo in an animal model.

OBJECTIVES: The aim of this study is to evaluate long-term in vivo stability of dental implants stabilized at time of placement in oversized osteotomies with a novel, self-setting, mineral-organic bone adhesive. MATERIALS/METHODS: Canine (26) mandibular teeth were removed, and three oversized osteotomies prepared bilaterally. Implants were placed with either adhesive, particulate xenograft, or with blood clot filling the implant/osteotomy gaps. Removal torque and histology were assessed. RESULTS: The adhesive provided significant and clinically relevant immediate implant stability of 22.2 N-cm (95% CI 5.3; 39.0), which continued throughout the early postoperative course and persisted through the nine- (155 N-cm 95% CI 113; 197) and 12-month (171 N-cm 95% CI 134.2; 209.4) time points. This is in comparison with the blood clot of 1.4 N-cm (95% CI 0.7; 2.1), 128.6 N-cm (95% CI 66.8; 190.4), and 140.7 N-cm (95% CI 78.8; 202.5) and particulate xenograft, 1.3 N-cm (95% CI 0.6; 2.0), 132.1 N-cm (95% CI 94.5; 169.7), and 101.5 (95% CI 59.5; 143.5), respectively. Histological examination shows the adhesive establishes intimate contact with the implant and bony walls and is replaced with new bone without compromising stability. Soft tissue does not penetrate the adhesive, and marginal bone/biomaterial level is maintained. Control sites filled with xenograft or blood clot heal with reduced bone levels, and in some cases, xenograft particles were encapsulated in connective tissue. CONCLUSIONS: Implants placed in oversized osteotomies and lacking primary stability can be stabilized at placement with a novel, highly osteoconductive, and resorbable adhesive. Gradual replacement of the biomaterial allows osseointegration without loss of stability through 12 months of follow-up. This novel adhesive has the potential to stabilize implants placed in sites with inadequate bony support.

Amyloid clearance defect in ApoE4 astrocytes is reversed by epigenetic correction of endosomal pH.

Endosomes have emerged as a central hub and pathogenic driver of Alzheimer's disease (AD). The earliest brain cytopathology in neurodegeneration, occurring decades before amyloid plaques and cognitive decline, is an expansion in the size and number of endosomal compartments. The strongest genetic risk factor for sporadic AD is the ε4 allele of Apolipoprotein E (ApoE4). Previous studies have shown that ApoE4 potentiates presymptomatic endosomal dysfunction and defective endocytic clearance of amyloid beta (Aß), although how these two pathways are linked at a cellular and mechanistic level has been unclear. Here, we show that aberrant endosomal acidification in ApoE4 astrocytes traps the low-density lipoprotein receptor-related protein (LRP1) within intracellular compartments, leading to loss of surface expression and Aß clearance. Pathological endosome acidification is caused by ε4 risk allele-selective down-regulation of the Na+/H+ exchanger isoform NHE6, which functions as a critical leak pathway for endosomal protons. In vivo, the NHE6 knockout (NHE6KO) mouse model showed elevated Aß in the brain, consistent with a causal effect. Increased nuclear translocation of histone deacetylase 4 (HDAC4) in ApoE4 astrocytes, compared with the nonpathogenic ApoE3 allele, suggested a mechanistic basis for transcriptional down-regulation of NHE6. HDAC inhibitors that restored NHE6 expression normalized ApoE4-specific defects in endosomal pH, LRP1 trafficking, and amyloid clearance. Thus, NHE6 is a downstream effector of ApoE4 and emerges as a promising therapeutic target in AD. These observations have prognostic implications for patients who have Christianson syndrome with loss of function mutations in NHE6 and exhibit prominent glial pathology and progressive hallmarks of neurodegeneration.

Evaluation and comparison of histologic changes and implant survival in extraction sites immediately grafted with two different xenografts: A randomized clinical pilot study.

OBJECTIVES: The purpose of this prospective, single-center randomized pilot study was to histologically evaluate and compare vital bone development in premolar and molar-extraction sites grafted with two different bovine-derived xenografts. The secondary outcome of interest was implant survival in the grafted sites. MATERIALS AND METHODS: Adult patients in need of at least two tooth extractions were enrolled. A paired design was used; each patient received at least one of each type of graft at different sites. Each extraction site was randomized to one of two xenograft treatment groups. A resorbable membrane was always placed, and primary intention soft tissue closure was achieved. Four months later, implants were placed and a trephine drill was used to remove bone cores for histologic and histomorphometric analysis. RESULTS: Sixteen patients with 40 extraction sites were enrolled; 20 sites were grafted with one type of xenograft and 20 with another. Mean patient age was 53.5 years, and 65% of patients were male. Evaluation of bone core samples taken from grafted sites showed no significant difference in the mean value of percentage of new bone formation between the different grafted sites (33.4% and 32.4%, p = .76). Cumulative implant survival was 97.5% at the 24-month follow-up visit. CONCLUSION: Within the limitations of this pilot study, no statistically significant differences in new bone growth between sites grafted with two different types of xenograft were found. Both graft materials promoted the formation of new bone and provided osseous support for implant placement after socket grafting.

Atypical Presentation of Crimean-Congo Hemorrhagic Fever as Ascending Paralysis with Rhabdomyolysis.

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by a arbovirus. It is asymptomatic in infected animals but a serious threat to the health of individuals. In human, it starts with nonspecific febrile diseases and progresses into severe hemorrhagic syndrome with high-casual fatality. Here, we report a case of CCHF with atypical presentation of ascending paralysis and rhabdomyolysis. HOW TO CITE THIS ARTICLE: Prasad HR, Sharma A, Kothari N, Vyas V, Goyal S. Atypical Presentation of Crimean-Congo Hemorrhagic Fever as Ascending Paralysis with Rhabdomyolysis. Indian J Crit Care Med 2020;24(2):143-144.

NHA2 promotes cyst development in an in vitro model of polycystic kidney disease.

KEY POINTS: Significant and selective up-regulation of the Na+ /H+ exchanger NHA2 (SLC9B2) was observed in cysts of patients with autosomal dominant polycystic kidney disease. Using the MDCK cell model of cystogenesis, it was found that NHA2 increases cyst size. Silencing or pharmacological inhibition of NHA2 inhibits cyst formation in vitro. Polycystin-1 represses NHA2 expression via Ca2+ /NFAT signalling whereas the dominant negative membrane-anchored C-terminal fragment (PC1-MAT) increased NHA2 levels. Drugs (caffeine, theophylline) and hormones (vasopressin, aldosterone) known to exacerbate cysts elicit NHA2 expression. Taken together, the findings reveal NHA2 as a potential new player in salt and water homeostasis in the kidney and in the pathogenesis of polycystic kidney disease. ABSTRACT: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up-regulation of NHA2, a member of the SLC9B family of Na+ /H+ exchangers, that correlated with cyst size and disease severity in ADPKD patients. Using three-dimensional cultures of MDCK cells to model cystogenesis in vitro, we showed that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store-dependent and -independent pathways, whereas reciprocal activation of Ca2+ influx by the dominant negative membrane-anchored C-terminal tail fragment of PC1 elevated NHA2. We showed that NHA2 is a target of Ca2+ /NFAT signalling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observed robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up-regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for polycystic kidney disease and reveal a potential new regulator of transepithelial salt and water transport in the kidney.

Histone deacetylase-mediated regulation of endolysosomal pH.

The pH of the endolysosomal system is tightly regulated by a balance of proton pump and leak mechanisms that are critical for storage, recycling, turnover, and signaling functions in the cell. Dysregulation of endolysosomal pH has been linked to aging, amyloidogenesis, synaptic dysfunction, and various neurodegenerative disorders, including Alzheimer's disease. Therefore, understanding the mechanisms that regulate luminal pH may be key to identifying new targets for managing these disorders. Meta-analysis of yeast microarray databases revealed that nutrient-limiting conditions inhibited the histone deacetylase (HDAC) Rpd3 and thereby up-regulated transcription of the endosomal Na+/H+ exchanger Nhx1, resulting in vacuolar alkalinization. Consistent with these findings, Rpd3 inhibition by the HDAC inhibitor and antifungal drug trichostatin A induced Nhx1 expression and vacuolar alkalinization. Bioinformatics analysis of Drosophila and mouse databases revealed that caloric control of the Nhx1 orthologs DmNHE3 and NHE6, respectively, is also mediated by HDACs. We show that NHE6 is a target of the transcription factor cAMP-response element-binding protein (CREB), a known regulator of cellular responses to low-nutrient conditions, providing a molecular mechanism for nutrient- and HDAC-dependent regulation of endosomal pH. Of note, pharmacological targeting of the CREB pathway to increase NHE6 expression helped regulate endosomal pH and correct defective clearance of amyloid Aß in an apoE4 astrocyte model of Alzheimer's disease. These observations from yeast, fly, mouse, and cell culture models point to an evolutionarily conserved mechanism for HDAC-mediated regulation of endosomal NHE expression. Our insights offer new therapeutic strategies for modulation of endolysosomal pH in fungal infection and human disease.

Sinus augmentation using rhBMP-2/ACS in a mini-pig model: Influence of an adjunctive ceramic bone biomaterial.

BACKGROUND: Recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge carrier (rhBMP-2/ACS) has been shown to support significant bone formation when used to augment the maxillary sinus for implant dentistry. Nevertheless, bone biomaterials have been suggested to extend rhBMP-2/ACS with limited support of the merits of such approaches. OBJECTIVES: To evaluate local bone formation/dental implant osseointegration following implantation of rhBMP-2/ACS combined with a ceramic bone biomaterial using a mini-pig sinus augmentation model. METHODS: Twelve adult Göttingen mini-pigs received rhBMP-2/ACS (rhBMP-2 adjusted to 0.43 mg/cc) alone or combined with an off-the-shelf biphasic ceramic (15%/85% HA/ß-TCP) biomaterial at 3:1, 1:1 and 1:3 ratios randomized to contra-lateral maxillary sinus sites yielding rhBMP-2/ACS fractions of 100%, 75%, 50% and 25%, respectively. A 4-cc implant volume was used for all sites. Two threaded dental implants (ø4.0 × 11.5 mm) were placed at each site. The animals were euthanized at 8 weeks for histologic analysis. RESULTS: Surgical execution and healing were generally uneventful, infraorbital local swelling was observed in all animals until suture removal. rhBMP-2/ACS combined with the ceramic biomaterial did not significantly enhance local bone formation (range 9.0 ± 1.5 to 9.7 ± 2.1 mm) compared with rhBMP-2/ACS alone (8.6 ± 1.1 mm; p > 0.05). Variations in rhBMP-2/ACS to ceramic matrix ratios yielding rhBMP-2 doses approximating 0.4, 0.9, 1.3 and 1.7 mg/sinus did not appreciably influence bone formation/osseointegration. CONCLUSIONS: Whereas rhBMP-2/ACS supports significant bone formation/osseointegration in the mini-pig sinus augmentation model and thus appears an effective alternative for sinus augmentation procedures, adding a ceramic biomaterial to rhBMP-2/ACS does not produce meaningful biological advantages.

Histomorphometric Comparison of 3 Osteotomy Techniques.

PURPOSE: This pilot study compares the histomorphometric structure of osteotomy preparation through standard extraction drilling (SD), Summers osteotomes (SO), and a new method of nonextraction drilling called osseodensification (OD). METHOD AND MATERIALS: Fresh porcine tibia plateau was used as the surgical specimen. Three preparation methods (N = 6 for each) were used to prepare 18 osteotomies according to manufacturer protocols. Eighteen tapered screw-vent (4.7 × 13 mm) implants were placed. After osteotomy preparation and implant placement, all porcine tibias were placed in 10% formalin solution in preparation for histological staining and sectioning. Histomorphometric analysis of all samples was performed to compare immediate bone-to-implant contact (BIC) and the percentage of bone volume within a 2-mm zone surrounding the implant. RESULTS: OD achieved 60.3% BIC, SO 40.7% BIC, and standard extraction drilling (SD) 16.3% BIC. The percentage of bone volume in the surrounding 2-mm width from the implant body using the same area units per sample was found to be greatest for OD. CONCLUSION: This study demonstrated that osteotomy preparation can influence both BIC and percentage of bone volume around the implant.

Postmortem evaluation of mandibular implant-supported fixed complete denture after 30 years of service.

A postmortem evaluation of a 5-implant-supported mandibular fixed complete denture that had successfully opposed a maxillary conventional complete denture for 30 years was undertaken. Before embalming, radiographs, implant stability measurements, push-in failure load tests, and histomorphometric analyses were performed on the implants and the mandible. Evaluation of this cadaver suggests that an edentulous mandible restored with an implant-supported fixed prosthesis can function successfully for over 30 years with few complications.

Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells.

Calcification of the breast is often an outward manifestation of underlying molecular changes that drive carcinogenesis. Up to 50% of all non-palpable breast tumors and 90% of ductal carcinoma in situ present with radiographically dense mineralization in mammographic scans. However, surprisingly little is known about the molecular pathways that lead to microcalcifications in the breast. Here, we report on a rapid and quantitative in vitro assay to monitor microcalcifications in breast cancer cell lines, including MCF7, MDA-MB-231, and Hs578T. We show that the Secretory Pathway Ca2+ -ATPases SPCA1 and SPCA2 are strongly induced under osteogenic conditions that elicit microcalcifications. SPCA gene expression is significantly elevated in breast cancer subtypes that are associated with microcalcifications. Ectopic expression of SPCA genes drives microcalcifications and is dependent on pumping activity. Conversely, knockdown of SPCA expression significantly attenuates formation of microcalcifications. We propose that high levels of SPCA pumps may initiate mineralization in the secretory pathway by elevating luminal Ca2+ . Our new findings offer mechanistic insight and functional implications on a widely observed, yet poorly understood radiographic signature of breast cancer.

Sinus augmentation using a mini-pig model: Effect of ceramic and allogeneic bone biomaterials.

BACKGROUND: Present clinical practice broadly relies on off-the-shelf allogeneic, xenogeneic or synthetic bone biomaterials in support of sinus augmentation. Also, recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge carrier (rhBMP-2/ACS) has been shown to support clinically relevant bone formation when used to augment the maxillary sinus. OBJECTIVES: To evaluate local bone formation/dental implant osseointegration following implantation of two particulate bone biomaterials using the mini-pig sinus augmentation model. METHODS: Nine adult Göttingen mini-pigs were used for evaluation of a biphasic ceramic (15%/85% HA/ß-TCP) and an allogeneic mineralized bone biomaterial. Treatments randomized to contralateral sinus sites included sham-surgery (control) and biomaterials. Two threaded dental implants (ø4.0 × 11.5 mm) were placed at each sinus site. The animals were euthanized at 8 weeks for histologic analysis. RESULTS: Execution of the surgical protocol and healing was unremarkable. Limited infraorbital swelling was observed until suture removal. The biphasic ceramic and allogeneic bone biomaterials produced significantly increased bone formation (5.2 ± 1.9 mm and 4.9 ± 1.6 mm vs. 2.6 ± 0.5 mm, p < 0.05) and osseointegration (18.0 ± 6.0% and 25.1 ± 18.2% vs. 10.1 ± 8.0%, p < 0.05) over the sham-surgery control. No significant differences were observed between biomaterials. CONCLUSIONS: Implantation of biphasic ceramic or allogeneic bone biomaterials enhances bone formation in the mini-pig maxillary sinus, however, dental implant bone support is incomplete resulting in overall limited osseointegration.

The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease.

Early intervention may be key to safe and effective therapies in patients with Alzheimer disease. Endosomal dysfunction is an early step in neurodegeneration. Endosomes are a major site of production of Aß peptide from the processing of amyloid precursor protein (APP) by clipping enzymes (ß- and γ-secretases). The ß-secretase enzyme BACE1 requires acidic lumen pH for optimum function, and acid pH promotes Aß aggregation. The Na(+)/H(+) exchanger NHE6 provides a leak pathway for protons, limiting luminal acidification by proton pumps. Like APP, NHE6 expression was induced upon differentiation of SH-SY5Y neuroblastoma cells and localized to an endosomal compartment. Therefore, we investigated whether NHE6 expression altered APP localization and processing in a stably transfected cell culture model of human APP expression. We show that co-expression with NHE6 or treatment with the Na(+)/H(+) ionophore monensin shifted APP away from the trans-Golgi network into early and recycling endosomes in HEK293 cells. NHE6 alkalinized the endosomal lumen, similar to monensin, and significantly attenuated APP processing and Aß secretion. In contrast, Aß production was elevated upon NHE6 knockdown. We show that NHE6 transcript and protein levels are lowered in Alzheimer brains relative to control. These findings, taken together with emerging genetic evidence linking endosomal Na(+)/H(+) exchangers with Alzheimer disease, suggest that proton leak pathways may regulate Aß generation and contribute to disease etiology.

Proteome-Wide Profiling of Targets of Cysteine reactive Small Molecules by Using Ethynyl Benziodoxolone Reagents.

In this study, we present a highly efficient method for proteomic profiling of cysteine residues in complex proteomes and in living cells. Our method is based on alkynylation of cysteines in complex proteomes using a "clickable" alkynyl benziodoxolone bearing an azide group. This reaction proceeds fast, under mild physiological conditions, and with a very high degree of chemoselectivity. The formed azide-capped alkynyl-cysteine adducts are readily detectable by LC-MS/MS, and can be further functionalized with TAMRA or biotin alkyne via CuAAC. We demonstrate the utility of alkynyl benziodoxolones for chemical proteomics applications by identifying the proteomic targets of curcumin, a diarylheptanoid natural product that was and still is part of multiple human clinical trials as anticancer agent. Our results demonstrate that curcumin covalently modifies several key players of cellular signaling and metabolism, most notably the enzyme casein kinase I gamma. We anticipate that this new method for cysteine profiling will find broad application in chemical proteomics and drug discovery.

De novo bone regeneration in human extraction sites using recombinant human bone morphogenetic protein-2/ACS: a clinical, histomorphometric, densitometric, and 3-dimensional cone-beam computerized tomographic scan evaluation.

INTRODUCTION: To preserve alveolar bone after extractions, it is important to graft socket sites to prevent bone loss from repair and remodeling. OBJECTIVE: The objective of this case series was to assess the clinical, densitometric, and histomorphometric results from extraction sockets treated with recombinant human bone morphogenetic protein (rhBMP-2/ACS). STUDY DESIGN: After extraction and socket debridement, INFUSE (rhBMP-2) on absorbable sponges was placed over each socket. After 4 months, 3-dimensional cone-beam computerized tomographic (CT) scans were taken. Trephined bone cores were taken as the first step in the implant site osteotomy and submitted for histomorphometric analysis. RESULTS: Histomorphometric analysis showed a mean of 49.6% vital bone with a SD of 10.8%. CT scans showed mean density of 510.6 Hounsfield units. CONCLUSIONS: Use of INFUSE in socket preservation surgery results in adequate de novo bone formation to support ideal implant placement after 4 months.