Development of surface water exposure scenarios for risk assessment of pesticides in Korea.

Surface water exposure scenarios used in the risk assessment of Korea's aquatic ecosystems, were developed to represent the 90th percentile pesticide exposure situation as a part of the country's pesticide registration procedure. The scenarios are used to estimate the pesticide concentration in the water of a rice paddy and small streams for three protection goals: (i) mudfish in rice paddies, (ii) the aquatic ecosystem of small streams located near rice paddies, and (iii) the aquatic ecosystem of small streams located near fruit orchards. The scenarios were derived taking into account major exposure routes, such as spray drift, runoff, and drainage. The scenarios were parameterized for appropriate models including the pesticide root zone model (PRZM) and the toxic substances in surface waters model (TOXSWA). A total of 17 pesticide compounds and 28 formulated products were selected to test the risk assessment using the developed scenarios. The simulated predicted environmental concentrations (PECs) fully reflected a) the exposure routes for each protection goal b) the use patterns of the products c) physicochemical properties of the pesticides, and d) meteorological conditions of Korea. However, while assessing the risks for aquatic organisms we observed that for most of the selected pesticides the calculated exposure concentrations were higher than the regulatory acceptable concentration (RAC). To implement the exposure scenarios and models for pesticide authorization in Korea, further research on the RACs is needed. We also recommend studies to develop a higher-tier model and risk-mitigation measures that can be applied to the Korean situation.

Intra-articular delivery of synovium-resident mesenchymal stem cells via BMP-7-loaded fibrous PLGA scaffolds for cartilage repair.

Delivery of synovium-resident mesenchymal stem cells (synMSCs) to cartilage defect site might provide a novel therapeutic modality for treatment of articular cartilage diseases. However, low isolation efficiency of synMSCs limits their therapeutic application. Niche-preserving non-enzymatic isolation of synMSCs was firstly attempted by employing micro-organ culture system based on recapitulating tissue-specific homeostasis ex vivo. The isolated synMSCs retained superior long-term growth competency, proliferation and chondrogenic potential to bone marrow-derived MSCs (BMSCs). It was noted that synMSCs demonstrated 9-fold increase in cartilaginous micro-tissue formation and 13-fold increase in sulfated proteoglycans deposition compared to BMSCs. For delivery of synMSCs, fibrous PLGA scaffolds were specifically designed for full-thickness osteochondral defects in rabbits. The scaffolds provided effective micro-environment for growth and host-integration of synMSCs. Combined delivery of synMSCs with bone morphogenetic proteins-7 (BMP-7) was designed to achieve synergistic therapeutic efficacy. BMP-7-loaded PLGA nanoparticles electrosprayed onto the scaffolds released BMP-7 over 2 weeks to conform with its aimed role in stimulating early stage endochondral ossification. Scaffold-supported combined administration of synMSCs with BMP-7 resulted in high proteoglycan and collagen type II induction and thick hyaline cartilage formation. Intra-articular co-delivery of synMSCs with BMP-7 via fibrous PLGA scaffolds may be a promising therapeutic modality for articular cartilage repair.

Structural and biochemical analyses reveal ubiquitin C-terminal hydrolase-L1 as a specific client of the peroxiredoxin II chaperone.

Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.

The Protein Kinase A Pathway Regulates Zearalenone Production by Modulating Alternative ZEB2 Transcription.

Zearalenone (ZEA) is an estrogenic mycotoxin that is produced by several Fusarium species, including Fusarium graminearum. One of the ZEA biosynthetic genes, ZEB2, encodes two isoforms of Zeb2 by alternative transcription, forming an activator (Zeb2L-Zeb2L homooligomer) and an inhibitor (Zeb2L-Zeb2S heterodimer) that directly regulate the ZEA biosynthetic genes in F. graminearum. Cyclic AMP-dependent protein kinase A (PKA) signaling regulates secondary metabolic processes in several filamentous fungi. In this study, we investigated the effects of the PKA signaling pathway on ZEA biosynthesis. Through functional analyses of PKA catalytic and regulatory subunits (CPKs and PKR), we found that the PKA pathway negatively regulates ZEA production. Genetic and biochemical evidence further demonstrated that the PKA pathway specifically represses ZEB2L transcription and also takes part in posttranscriptional regulation of ZEB2L during ZEA production. Our findings reveal the intriguing mechanism that the PKA pathway regulates secondary metabolite production by reprograming alternative transcription.

Regioselective Nucleophilic Functionalization of Antiaromatic Nickel(II) Norcorroles.

Treatment of antiaromatic nickel(II) norcorrole with potassium cyanide provided nickel(II) 3-cyanonorcorrole with perfect regioselectivity without the help of a catalyst. The reaction of the nickel(II) norcorrole with phenol or thiophenol in the presence of a base also yielded substitution products. The antiaromatic 16π conjugation system in the norcorrole core was preserved in the functionalized products. Introduction of phenylthio groups significantly decreased the HOMO-LUMO gap and enhanced the near IR absorption property.

Peroxisome proliferator-activated receptor γ upregulates galectin-9 and predicts prognosis in intestinal-type gastric cancer.

The importance of PPARγ (peroxisome proliferator-activated receptor γ) in gastric cancer (GC) is unclear. We investigated the role of PPARγ in GC cell lines and an animal model, and its prognostic significance of PPARγ in GC patients. We controlled PPARγ and galectin-9 expression by using siRNAs and lentiviral constructs. Interaction between PPARγ and galectin-9 was evaluated using luciferase and chromatin immunoprecipitation assays. PPARγ expression in GCs was determined by immunohistochemical staining of tissue microarrays and survival analysis was done. Overexpression of PPARγ was accompanied by increased galectin-9. Enhanced PPARγ or galectin-9 expression increased E-cadherin expression; decreased expression of N-cadherin, fibronectin, snail, twist and slug and reduced cell invasion and migration. PPARγ bound to the galectin-9 promoter region. Galectin-9 activity increased in PPARγ-overexpressing cells but decreased in PPARγ siRNA-treated cells. In a zebrafish xenograft model, the number of migrated cancer cells and number of fish with AGS cells in the tail vein were reduced in PPARγ-overexpressing GC cells. PPARγ was expressed in 462 of the 688 patients (69.2%) with GC. In 306 patients with intestinal-type GC, those with PPARγ-positive tumors had lower overall and cancer-specific mortalities than those with PPARγ-negative tumors. PPARγ expression was an independent prognostic factor for overall and GC-specific mortality in patients with intestinal-type GC (adjusted hazard ratio, 0.42; 95% CI, 0.22-0.81). PPARγ inhibits cell invasion, migration and epithelial-mesenchymal transition through upregulation of galectin-9 in vitro and in vivo.

A 3-pyridyl-5,15-diazaporphyrin nickel(II) complex as a bidentate metalloligand for transition metals.

3-Pyridyl-5,15-diazaporphyrin nickel(II) serves as a bidentate metalloligand for platinum(II), ruthenium(II), and rhenium(I) metal centers. Single-crystal X-ray diffraction analysis of these metal complexes unambiguously reveals the presence of a dative bond between the outer metal center and the meso-nitrogen atom. The UV/Vis absorption spectra of the complexes show substantially red-shifted bands which are perturbed by outer-metal coordination. This is due to the contribution of metal-to-ligand charge transfer interactions.

Comparison of digene hybrid capture 2, GeneMatrix PapilloScreen, and a PCR sequencing assay in detecting high-risk and probable high-risk oncogenic HPV genotypes in specimens from Korean women.

Most cervical cancers are caused by 15 high-risk (HR) and three probable high-risk (pHR) oncogenic types of human papillomavirus (HPV). However, current commercial HR HPV screening test products do not include pHR HPV genotypes. Recently, PapilloScreen has been developed to detect the 15 HR and three pHR HPV types. In this study, we evaluated the concordance levels and clinical performance of Hybrid Capture 2 (HC2), PapilloScreen, and a PCR sequencing assay in detecting HR and pHR HPV. The PapilloScreen (96.8 %) and PCR sequencing assay (96.8 %) demonstrated higher sensitivity than HC2 (80.7 %) for detecting HR and pHR HPV. The three assays showed similar specificities and positive or negative predictive values. The concordance levels were 86.5 % (κ = 0.68) and 86.5 % (κ = 0.67) between HC2 and PapilloScreen and between HC2 and PCR sequencing, respectively. A near-perfect concordance was observed between PapilloScreen and PCR sequencing (97.8 %, κ = 0.95). Overall, the agreement between the three assays suggests that the results obtained by the HC2 assay are more often discordant (12.6 %) than the PCR-based tests. In conclusion, PapilloScreen is highly sensitive for detecting high-grade CIN or cervical cancer. The PapilloScreen assay should be considered an accurate and sensitive method for detecting HR and pHR HPV infections and an epidemiological tool for prevalence studies as well as early diagnosis and intervention in HR and pHR HPV infections.

MicroRNA 135a suppresses lymph node metastasis through down-regulation of ROCK1 in early gastric cancer.

MicroRNAs (miRNAs) play a critical role in gastric cancer progression and metastasis. This study investigated the role of miRNA-135a in early gastric cancer (EGC) including lymph node (LN) metastasis. We examined the correlation between miRNA-135a expression and clinical outcomes in 59 patients who underwent surgery for EGC. Using gastric cancer cell lines, we performed functional and target gene analyses. miRNA-135a expression was down-regulated in 33.9% of patients. These patients showed a significantly more advanced stage (TNM stage ≥ IB, 35.0% vs. 12.8%, p = 0.045) and higher rate of LN metastasis (30.0% vs. 5.1%, p = 0.014) than those with up-regulation of miRNA-135a expression. In a multivariate analysis, down-regulation of miRNA-135a was an independent risk factor for LN metastasis (adjusted odds ratio, 8.04; 95% confidence interval, 1.08-59.81; p = 0.042). Functional analyses using gastric cancer cell lines showed that miRNA-135a suppressed cell viability, epithelial-mesenchymal transition, cell invasion, and migration. ROCK1 was a target of miRNA-135a and its expression was inversely correlated to that of miRNA-135a. ROCK1 expression was significantly increased in EGC patients with LN metastasis than in those without LN metastasis. Our results confirm the tumor-suppressive role of miRNA-135a, and demonstrate its role in LN metastasis in EGC. miRNA-135a and its target gene ROCK1 may be novel therapeutic and prognostic targets for EGC.

Near-IR absorbing nickel(II) porphyrinoids prepared by regioselective insertion of silylenes into antiaromatic nickel(II) norcorrole.

The treatment of an antiaromatic norcorrole Ni(II) complex with a kinetically stabilized silylene provided ring-expansion products in excellent yields through the highly regio- and stereoselective insertion into the ß-ß pyrrolic CC bonds. The resultant Ni(II) porphyrinoid monoinsertion product exhibited relatively strong near-IR absorption bands due to the small HOMO-LUMO gap in spite of the disrupted cyclic π-conjugation by the silicon atom.

The effect of RNAi silencing of p62 using an osmotic polysorbitol transporter on autophagy and tumorigenesis in lungs of K-rasLA1 mice.

Treating cancer patients by conventional chemotherapy to achieve prolonged survival still remains complicated. Autophagy is a topic of considerable interest in recent times, as it may contribute greatly to tumor suppression. Recent studies indicate that autophagy-deficient cells accumulate high levels of p62, an ubiquitin-binding scaffold protein, involved greatly in tumorigenesis. Here, we synthesized an osmotically active polysorbitol-mediated transporter (PSMT) to downregulate p62 using an RNAi strategy and described the mechanism of how p62 silencing using PSMT/siRNA p62 system activates autophagy and contributes to tumor suppression in the lungs of K-ras(LA1) mice. Downregulation of p62 by PSMT/siRNA p62 activated autophagy confirmed by the formation of autophagosomes and swelling of Golgi apparatus with a decreasing level of GM130, a cis-Golgi matrix protein. Activation of osmotic PSMT-mediated autophagy remarkably reduced the size and number of tumors by suppressing proliferative cell nuclear antigen, cluster of differentiation 31, and vascular endothelial growth factor levels. Furthermore, an increase in apoptosis was observed in the lungs of PSMT/siRNA p62-delivered K-ras(LA1) mice.

Extremely increased serum carbohydrate antigen 19-9 levels caused by new or resistant infections to previous antibiotics in chronic lung diseases.

In this paper, we describe 72-year-old female patient without evidence of malignant disease presented with significantly elevated serum carbohydrate antigen (CA) 19-9 levels by respiratory infections. She was diagnosed with respiratory infections due to Mycobacterium avium complex and Pseudomonas aeruginosa. The serum CA 19-9 levels remarkably increased (1,453-5,300 U/mL; reference range, <37 U/mL) by respiratory infection and abruptly decreased (357-534 U/mL) whenever infection was controlled by specific treatments. This case suggests that serum CA 19-9 levels may be used as a diagnostic marker to indicate new or resistant infections to previous antibiotics in chronic lung diseases without significant changes in chest X-ray findings.

Overexpression of beclin1 induced autophagy and apoptosis in lungs of K-rasLA1 mice.

Beclin1, as a key molecule in controlling autophagy pathway, can activate both cell survival and cell death pathway. As a role of autophagy in cancer progression remains controversial, introduction of beclin1 to the lungs of K-ras(LA1) mice was performed via inhalation. Prolonged autophagy activation was induced by repeated exposure of lentivirus-beclin1, total of 8 times (2 times/week, 4 weeks). By the time of sacrifice, lungs were collected and analyzed for the therapeutic efficacy. Total numbers of tumors on the surface and histopathological tumor progression were reduced in the lungs of K-ras(LA1) mice. Successful delivery of beclin1 induced autophagy and apoptosis in the target organ, which were confirmed by following features; increased autophagic vacuoles in the cytosol, increased number of mitochondria with decreased mitochondrial 12S RNA, and increased protein levels of mitochondria-related apoptosis. Markers for cell proliferation and angiogenesis, PCNA and VEGF, which used for prediction of cancer prognosis, were significantly reduced after introduction of beclin1. Taken together, the results indicate that autophagy regulating gene, beclin1, can be a potential target for lung cancer gene therapy.

Comparison of the clinical performance of restriction fragment mass polymorphism (RFMP) and Roche linear array HPV test assays for HPV detection and genotyping.

BACKGROUND: The need for accurate genotyping of human papillomavirus (HPV) infections is becoming increasingly important as HPV is the primary cause of cervical cancer worldwide. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry-based restriction fragment mass polymorphism (RFMP) assay provides accurate, broad-spectrum, high-throughput genotyping of HPV. OBJECTIVES: We evaluated the clinical performance of the RFMP assay compared to a commercially available Roche linear array HPV genotyping test (LA) for detecting and genotyping of HPV. STUDY DESIGN: The RFMP assay and the LA were compared for detecting and genotyping HPV among a cohort of 244 liquid-based cytology samples. RESULTS: Overall, 216 specimens (93.1%, κ = 0.86) generated concordant results for the presence or absence of high-risk HPV (HR-HPV) by the two assays. The RFMP assay and the LA assay generated concordant, compatible, and discordant genotyping results for 79.3, 9.9, and 10.8%, respectively. The diagnostic sensitivity and specificity of RFMP and LA for the cervical lesions of squamous cell carcinoma (SCC) were similar, at 92.9 and 85.0% (RFMP) and 92.9 and 83.8% (LA), respectively. In addition, the odds ratio for SCC with HR-HPV positivity estimated by the RFMP assay (73.7, 95% CI: 8.9-3173.3) was higher than the LA assay (67.0, 95% CI: 8.2-2887.0). CONCLUSIONS: The RFMP and the LA assays were highly comparable with regard to detection and genotyping analysis of HPV. The sensitivity and specificity of RFMP assay for the detection of HR-HPV in various levels of cervical lesions seems to be valuable in the monitoring of HPV-associated cervical cancer.

Aerosol delivery of lentivirus-mediated O-glycosylation mutant osteopontin suppresses lung tumorigenesis in K-ras (LA1) mice.

BACKGROUND: Osteopontin (OPN) is a secreted glycophosphoprotein that has been implicated in the regulation of cancer development. The function of OPN is primarily regulated through post-translational modification such as glycosylation. As yet, however, the relationship between OPN glycosylation and lung cancer development has not been investigated. In this study, we addressed this issue by studying the effect of a triple mutant (TM) of OPN, which is mutated at three O-glycosylation sites, on lung cancer development in K-ras (LA1) mice, a murine model for human non-small cell lung cancer. METHODS: Aerosolized lentivirus-based OPN TM was delivered into the lungs of K-ras (LA1) mice using a nose-only-inhalation chamber 3 times/wk for 4 wks. Subsequently, the effects of repeated delivery of OPN TM on lung tumorigenesis and its concomitant OPN-mediated signaling pathways were investigated. RESULTS: Aerosol-delivered OPN TM inhibited lung tumorigenesis. In addition, the OPN-mediated Akt signaling pathway was inhibited. OPN TM also decreased NF-κB activity and the phosphorylation of 4E-BP1, while facilitating apoptosis in the lungs of K-ras (LA1) mice. CONCLUSIONS: Our results show that aerosol delivery of OPN TM successfully suppresses lung cancer development in the K-ras (LA1) mouse model and, therefore, warrant its further investigation as a possible therapeutic strategy for non-small cell lung cancer.

Acceleration of gastric tumorigenesis through MKRN1-mediated posttranslational regulation of p14ARF.

BACKGROUND: We investigated whether Makorin ring finger protein 1 (MKRN1), an E3 ligase, affects p14ARF-associated cellular senescence and tumorigenesis by posttranslational modification in gastric tumorigenesis. METHODS: A link between MKRN1 and ARF was examined in MKRN1 null mouse embryonic fibroblasts (MEFs) and in human fibroblasts and gastric cancer cells by silencing MKRN1 using small interfering RNA (siRNA) and short hairpin RNA (shRNA). Ubiquitination and proteasomal degradation assays were used to assess p14ARF degradation associated with MKRN1. MKRN1 and p14ARF expression levels were analyzed with immunohistochemistry in malignant and normal tissues from gastric cancer patients and with χ(2) tests. The tumor growth of gastric cancer cells stably expressing MKRN1 shRNA, p14ARF shRNA, or both was examined in mouse xenograft models (n = 4-6) and analyzed with unpaired t tests. All statistical tests were two-sided. RESULTS: MKRN1 knockout MEFs exhibited premature senescence and growth retardation with increased p19ARF protein expression. Similar results were obtained for human fibroblasts or gastric cancer cell lines by MKRN1 knockdown. Biochemical analyses confirmed that MKRN1 targets p14ARF for ubiquitination and subsequent proteasome-dependent degradation. A statistically significant association was shown between MKRN1 overexpression and p14ARF underexpression (P = .016). Xenograft analyses using p53-functional AGS or -dysfunctional SNU601 cells displayed statistically significant tumor growth retardation by silencing MKRN1, which was reversed under depletion of p14ARF (AGS cells, MKRN1 knockdown tumors vs MKRN1 and p14ARF knockdown tumors: 164.6 vs 464.8mm(3), difference = 300.2mm(3), 95% CI = 189.1 to 411.3mm(3), P < .001). CONCLUSIONS: We demonstrated that MKRN1 functions as a novel E3 ligase of p14ARF and that it potentially regulates cellular senescence and tumorigenesis in gastric cancer.

The role of osmotic polysorbitol-based transporter in RNAi silencing via caveolae-mediated endocytosis and COX-2 expression.

Polymeric diversity allows us to design gene carriers as an alternative to viral vectors, control cellular uptake, target intracellular molecules, and improve transfection and silencing capacity. Recently, we developed a polysorbitol-based osmotically active transporter (PSOAT), which exhibits several interesting mechanisms to accelerate gene delivery into cells. Herein, we report the efficacy of using the PSOAT system for small interfering RNA (siRNA) delivery and its specific mechanism for cellular uptake to accelerate targeted gene silencing. We found that PSOAT functioned via a caveolae-mediated uptake mechanism due to hyperosmotic activity of the transporter. Moreover, this selective caveolae-mediated endocytosis of the polyplexes (PSOAT/siRNA) was regulated coincidently with the expression of caveolin (Cav)-1 and cyclooxygenase (COX)-2. Interestingly, COX-2 expression decreased dramatically over time due to degradation by the constant expression of Cav-1, as confirmed by high COX-2 expression after the inhibition of Cav-1, suggesting that PSOAT-mediated induction of Cav-1 directly influenced the selective caveolae-mediated endocytosis of the polyplexes. Furthermore, COX-2 expression was involved in the initial phase for rapid caveolae endocytic uptake of the particles synergistically with Cav-1, resulting in accelerated PSOAT-mediated target gene silencing.

Aerosol delivery of beclin1 enhanced the anti-tumor effect of radiation in the lungs of K-rasLA1 mice.

Radiotherapy alone has several limitations for treating lung cancer. Inhalation, a non-invasive approach for direct delivery of therapeutic agents to the lung, may help to enhance the therapeutic efficacy of radiation. Up-regulating beclin1, known as a tumor suppressor gene that plays a major role in autophagy, may sensitize tumors and lead to tumor regression in lungs of K-ras(LA1) lung cancer model mice. To minimize the side-effects of radiotherapy, fractionated exposures (five times, 24-h interval) with low dose (2 Gy) of radiation to the restricted area (thorax, 2 cm) were conducted. After sensitizing the lungs with radiation, beclin1, complexed with a nano-sized biodegradable poly(ester amine), was prepared and delivered into the murine lung via aerosol three times/week for four weeks. In a histopathological analysis, animals treated with beclin1 and radiation showed highly significant tumor regression and low progression to adenocarcinoma. An increase in the number of autophagic vacuoles and secondary lysosomes was detected. Dissociation of beclin1-bcl2 stimulated autophagy activation and showed a synergistic anti-tumor effect by inhibiting the Akt-mTOR pathway, cell proliferation and angiogenesis. The combination of radiation with non-invasive aerosol delivery of beclin1 may provide a prospect for developing novel therapy regimens applicable in clinics.

Gram-scale synthesis of nickel(II) norcorrole: the smallest antiaromatic porphyrinoid.

Small is beautiful: A ring-contracted sister of porphyrin, norcorrole, has been synthesized efficiently as a stable molecule by a nickel-templated strategy. The norcorrole complex is stable but exhibits a distinct antiaromatic character according to the Hückel rule. Oxidation of the norcorrole complex provides an aromatic oxacorrole complex.

High gene transfer by the osmotic polysorbitol-mediated transporter through the selective caveolae endocytic pathway.

Cationic polymers have been the subject of intense research as nonviral gene delivery systems due to several advantages in comparison with viral vectors. However, the nonsimultaneous combination of high transfection efficiency and low cytotoxicity of nonviral vectors for gene delivery has long been an issue for scientists looking into ways to deliver genes into cells. Toward this goal, we designed, synthesized, and evaluated a safe and accelerated gene transfer system through polysorbitol-mediated transporter (PSMT) based on sorbitol diacrylate (SDA) and low molecular weight polyethylenimine (LMW PEI). The PSMT formed stable complexes with plasmid DNA in serum. The nano sizes and spherical shapes of PSMT/DNA complexes are not toxic, even at a high concentration of PSMT. The higher transfection efficiency of PSMT compared to PEI 25K was observed both in vitro, despite the existence of many hydroxyl groups, and in vivo. These improvements presumably stem from the osmotic property of polysorbitol and endosomal buffer capacity of PEI in PSMT. Most importantly, we confirmed that the selective cavaeolae endocytic pathway played a role in high transfection efficiency by osmotic PSMT-mediated gene delivery. We propose that PSMT is a promising nonviral carrier for the effective gene delivery to cancer cells via synergistic effects derived from rapid cellular uptake through the caveolae endocytic pathway and a high endosomal buffering capacity.