[Geographic environment-related research advances on children's myopia: intraocular and environmental exposure factors and analytical methods].

Myopia has become a global phenomenon, transitioning into a significant public health issue of worldwide reach. The escalating prevalence of myopia may lead to an increase in the incidence of related complications, potentially resulting in irreversible vision damage for individuals. This not only causes considerable economic strain on societies but also poses a serious threat to vital sectors like national defense. This review outlines various external and internal exposure factors related to childhood myopia. It places particular focus on the analysis of the interaction between geographical environmental factors and internal exposure factors, and examines the limitations of applying traditional methods in studying the relationship between childhood myopia and geographical environmental factors. The paper also introduces two spatial regression methodologies based on frequency estimation and Bayesian estimation, summarizing their feasibility and merits when applied in the study of external exposure elements related to childhood myopia. Finally, it provides a fresh perspective on regional childhood myopia prevention strategies that are conscious of geographical environmental factors.

[Study of the urban-impact on microbial communities and their virulence factors and antibiotic resistance genomes in the Nandu River, Haikou].

Objective: To explore the changes in bacterial community structure, antibiotic resistance genome, and pathogen virulence genome in river water before and after the river flowing through Haikou City and their transmission and dispersal patterns and to reveal anthropogenic disturbance's effects on microorganisms and resistance genes in the aquatic environment. Methods: The Nandu River was divided into three study areas: the front, middle and rear sections from the upstream before it flowed through Haikou City to the estuary. Three sampling sites were selected in each area, and six copies of the sample were collected in parallel at each site and mixed for 3 L per sample. Microbial community structure, antibiotic resistance, virulence factors, and mobile genetic elements were analyzed through bioinformatic data obtained by metagenomic sequencing and full-length sequencing of 16S rRNA genes. Variations in the distribution of bacterial communities between samples and correlation of transmission patterns were analyzed by principal co-ordinates analysis, procrustes analysis, and Mantel test. Results: As the river flowed through Haikou City, microbes' alpha diversity gradually decreased. Among them, Proteobacteria dominates in the bacterial community in the front, middle, and rear sections, and the relative abundance of Proteobacteria in the middle and rear sections was higher than that in the front segment. The diversity and abundance of antibiotic resistance genes, virulence factors, and mobile genetic elements were all at low levels in the front section and all increased significantly after flow through Haikou City. At the same time, horizontal transmission mediated by mobile genetic elements played a more significant role in the spread of antibiotic-resistance genes and virulence factors. Conclusions: Urbanization significantly impacts river bacteria and the resistance genes, virulence factors, and mobile genetic elements they carry. The Nandu River in Haikou flows through the city, receiving antibiotic-resistant and pathogen-associated bacteria excreted by the population. In contrast, antibiotic-resistant genes and virulence factors are enriched in bacteria, which indicates a threat to environmental health and public health. Comparison of river microbiomes and antibiotic resistance genomes before and after flow through cities is a valuable early warning indicator for monitoring the spread of antibiotic resistance.

[Population structure of food-borne Staphylococcus aureus in China].

Objective: To understand the population structure of food-borne Staphylococcus (S.) aureus in China. Methods: Whole genome sequencing was used to analyze 763 food-borne S. aureus strains from 16 provinces in China from 2006 to 2020. Multilocus sequence typing (MLST), staphylococcal protein A gene (spa) typing, and staphylococcal chromosome cassettemec (SCCmec) typing were conducted, and minimum spanning tree based on ST types (STs) was constructed by BioNumerics 7.5 software. Thirty-one S. aureus strains isolated from imported food products were also included in constructing the genome phylogenetic tree. Results: A total of 90 STs (20 novel types) and 160 spa types were detected in the 763 S. aureus isolates. The 72 STs (72/90, 80.0%) were related to 22 clone complexes. The predominant clone complexes were CC7, CC1, CC5, CC398, CC188, CC59, CC6, CC88, CC15, and CC25, accounting for 82.44% (629/763) of the total. The STs and spa types in the predominant clone complexes changed over the years. The methicillin-resistant S. aureus (MRSA) detection rate was 7.60%, and 7 SCCmec types were identified. The ST59-t437-Ⅳa (17.24%, 10/58), ST239-t030-Ⅲ (12.07%, 7/58), ST59-t437-Ⅴb (8.62%, 5/58), ST338-t437-Ⅴb (6.90%, 4/58) and ST338-t441-Ⅴb (6.90%, 4/58) were the main types in MRSA strains. The genome phylogenetic tree had two clades, and the strains with the same CC, ST, and spa types clustered together. All CC7 methicillin sensitive S. aureus strains were included in Clade1, while 21 clone complexes and all MRSA strains were in Clade2. The MRSA strains clustered according to the SCCmec and STs. The strains from imported food products in CC398, CC7, CC30, CC12, and CC188 had far distances from Chinese strains in the tree. Conclusions: In this study, the predominant clone complexes of food-borne strains were CC7, CC1, CC5, CC398, CC188, CC59, CC6, CC88, CC15, and CC25, which overlapped with the previously reported clone complexes of hospital and community-associated strains in China, suggesting that close attention needs to be paid to food, a vehicle of pathogen transmission in community and food poisoning.

Evidence of neuroinflammation in fibromyalgia syndrome: a [ 18 F]DPA-714 positron emission tomography study.

ABSTRACT: This observational study aimed to determine whether individuals with fibromyalgia (FM) exhibit higher levels of neuroinflammation than healthy controls (HCs), as measured with positron emission tomography using [ 18 F]DPA-714, a second-generation radioligand for the translocator protein (TSPO). Fifteen women with FM and 10 HCs underwent neuroimaging. Distribution volume (V T ) was calculated for in 28 regions of interest (ROIs) using Logan graphical analysis and compared between groups using multiple linear regressions. Group (FM vs HC) was the main predictor of interest and TSPO binding status (high- vs mixed-affinity) was added as a covariate. The FM group had higher V T in the right postcentral gyrus ( b = 0.477, P = 0.033), right occipital gray matter (GM; b = 0.438, P = 0.039), and the right temporal GM ( b = 0.466, P = 0.042). The FM group also had lower V T than HCs in the left isthmus of the cingulate gyrus ( b = -0.553, P = 0.014). In the subgroup of high-affinity binders, the FM group had higher V T in the bilateral precuneus, postcentral gyrus, parietal GM, occipital GM, and supramarginal gyrus. Group differences in the right parietal GM were associated with decreased quality of life, higher pain severity and interference, and cognitive problems. In support of our hypothesis, we found increased radioligand binding (V T ) in the FM group compared with HCs in several brain regions regardless of participants' TSPO binding status. The ROIs overlapped with prior reports of increased TSPO binding in FM. Overall, increasing evidence supports the hypothesis that FM involves microglia-mediated neuroinflammation in the brain.

The role of alternative oxidase in plant hypersensitive response.

The innate immune system of plants is crucial in defining the fate of a plant cell during plant-pathogen interactions. This response is often accompanied by a hypersensitive reaction leading to the death of a plant cell and restricted pathogen growth. Plant mitochondria, in this case, play a key role by maintaining a balance between cell respiration and reactive oxygen species formation. One of the key features of the hypersensitive response is the shift of the normal plant respiratory pathway to a special 'alternative' pathway. Plants contain an enzyme, alternative oxidase, for maintaining metabolic homeostasis of the cell. This energy dissipating respiration provides a branch in normal respiration by using ubiquinone to form water and heat, thus maintaining the energy status of the cell. Alternative oxidase is thought to minimize production of reactive oxygen species and can also function in 'anti-apoptotic' machinery in plant cells. In this mini review, we briefly describe the alternative respiratory pathway and explain the role of alternative oxidase in important cellular processes, such as programmed cell death and the hypersensitive response.

Effect of different glucogenic to lipogenic nutrient ratios on rumen fermentation and bacterial community in vitro.

AIMS: This study was to investigate the effect of different ratios of glucogenic to lipogenic nutrients on rumen fermentation and the corresponding ruminal bacterial communities. METHODS AND RESULTS: Four diets, including glucogenic diet (G), lipogenic diet (L), two mixed diets: GL1 (G: L = 2 : 1) and GL2 (G:L = 1 : 2), served as substrates and were incubated with rumen fluid in vitro. The results revealed that the gas production, dry matter digestibility and propionate proportion were significantly increased by the G diet than others. The G diet increased the bacterial genera of Succinivibrionaceae_UCG_002, Succinivibrio, Selenomonas_1 and Ruminobacter but decreased some cellulolytic bacteria including the Eubacterium and several genera in family Ruminococcaceae than others. CONCLUSIONS: When the glucogenic nutrient was above 1/3 of the dietary energy source among the four diets, the in vitro incubation had a higher feed digestibility and lower acetate to propionate ratio. Bacterial genera, including Selenomonas, Succinivibrio, Ruminobacter, certain genera in Ruminococcaceae, Christensenellaceae_R-7_group and Eubacterium, were more sensitive to the glucogenic to lipogenic nutrients ratio. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides a new perspective about the effect of dietary glucogenic to lipogenic ingredient ratios on rumen metabolism by comparing end-products, gas production and bacterial composition via an in vitro technique.

The role of cyanide-resistant respiration in Solanum tuberosum L. against high light stress.

Cyanide-resistant respiration in potato mitochondria is an important pathway for energy dissipation. It can be activated by high light; however, it is unclear what roles cyanide-resistant respiration plays in the response to high light stress in potato. We designed a CRISPR vector for the functional gene StAOX of the potato cyanide-resistant respiratory pathway. Agrobacterium tumefaciens GV3101 was transformed into potato. Hydrogen peroxide level, MDA content, antioxidant activity and cyanide-resistant respiratory capacity of potato leaves under high light stress were determined. Photosynthetic efficiency and chlorophyll content were determined. In addition, the operation of the malate-oxaloacetate shuttle route and transcription level of photorespiration-related enzymes were also examined. The results showed that two base substitutions occurred at the sequencing target site on leaves of the transformed potato. Accumulation of ROS and increased membrane lipid peroxidation were detected in the transformed potato leaves and lower photosynthetic efficiency was observed. The transcription level of the malate-oxaloacetate shuttle route and photorespiration-related enzymes also significantly increased. These results indicate that the cyanide-resistant respiration is an important physiological pathway in potato in response to high light stress. It also suggests that plant cyanide-resistant respiration is closely related to photosynthesis. This implies the unexplored importance of plant cyanide-resistant respiration in plant photosynthesis, energy conversion and carbon skeleton formation.

AK5, a novel prognosis marker, inhibits apoptosis and promotes autophagy as well as proliferation in human gastric cancer.

OBJECTIVE: To explore the relationship between AK5 and gastric cancer. MATERIALS AND METHODS: The in situ levels of AK5 in the GC tissues from 255 patients were detected by immunohistochemistry (IHC). The correlation between AK5 expression and the clinicopathological parameters was analyzed by Pearson correlation, and the prognostic factors were identified by Cox regression analysis. The transcriptome data of 14 human GC cell lines deposited in the CCLE database were analyzed, and two lines were selected for functional studies. AK5 was knocked down in the AZ521 and MKN74 cells using siRNA, and their proliferation and apoptosis were evaluated by Cell Counting Kit-8 (CCK-8) assay and Annexin-V staining, respectively. In addition, the apoptosis and autophagy of the markers were detected by Western blotting. RESULTS: Patients expressing high AK5 levels in the tumor tissues had significantly shorter survival compared to low-expressing group. In addition, AK5 expression was associated with T stage and N stage and was an independent prognostic factor. AK5 knockdown in the AZ521 and MKN74 cells significantly inhibited proliferation and autophagy, and increased apoptosis. CONCLUSIONS: AK5 is a potential prognostic marker and therapeutic target for GC.

Compound climate events transform electrical power shortfall risk in the Pacific Northwest.

Power system reliability is sensitive to climate-driven variations in both energy demand and water availability, yet the combined effect of these impacts is rarely evaluated. Here we show that combined climate change impacts on loads and hydropower generation may have a transformative effect on the nature and seasonality of power shortfall risk in the U.S. Pacific Northwest. Under climate change, potential shortfall events occur more readily, but are significantly less severe in nature. A seasonal reversal in shortfall risk occurs: winter shortfalls are eradicated due to reduced building heating demands, while summer shortfalls multiply as increased peak loads for day-time cooling coincide with impaired hydropower generation. Many of these summer shortfalls go unregistered when climate change impacts on loads and hydropower dispatch are analyzed in isolation-highlighting an important role of compound events.

[Analysis on factors related to spontaneous reduction in twin pregnancy following assisted reproductive technology].

Objective: To explorer factors related to spontaneous reduction in twin pregnancy following assisted reproductive technology. Methods: 2 848 twin pregnant women with treatment of vitro fertilization-embryo transfer (IVF-ET) or intra cytoplasmic sperm injection (ICSI) cycles were enrolled at Assisted Reproductive Centre of the First Affiliated Hospital of Nanjing Medical University, Nanjing Maternity Hospital and Shengjing Hospital of China Medical University from January 2013 to December 2016 respectively. Basic features of subjects, relevant clinical indicators, factors of assisted reproductive therapy and pregnancy outcome were collected from clinical assisted reproductive technology management system. According to the pregnancy outcome, the subjects with spontaneous reduction were classified as case group (n=686), and those with normal twin birth were classified as control group (n=2 162). The features of subjects in the two groups were compared. Non-conditional logistics regression model was used to analyze the related factors of the occurrence of spontaneous reduction. Results: The age of case group and control group were (30.6±4.3) and (30.2±4.0) years old respectively. After the adjustment of male sterile factor, compared to the subjects with luteinizing hormone level on the day of human chorionic gonadotropin administration (HCG) <1.43 IU/L, OR (95%CI) of the subjects with value at 2.59-5.10 IU/L was1.62 (1.08-2.42).Compared to the subjects with number of transferred embryo as 1, OR (95%CI) of the subjects with value as 3 was 0.23 (0.07-0.74). Compared to the subjects with stage of transferred embryo as cleavage stage, OR (95%CI) of the subjects with blastula stage was 0.42 (0.27-0.67). Conclusion: Luteinizing hormone level on day of HCG, number and stage of embryo transfer are related factors to spontaneous reduction in twin pregnancy following assisted reproductive technology.

[Study of schistosomiasis transmission interruption based on intensive agriculture].

Objective To study the effect of schistosomiasis transmission interruption model based on intensive agriculture in hilly endemic areas, so as to provide the reference for the similar endemic areas. Methods Based on the development of intensive agriculture in Guanghan City, a comprehensive demonstration area of schistosomiasis control with measures such as new rural construction, hardening ditches, the adjustment of industrial structure and water remediation measures was constructed. Jinhua, Shiguan and Hongyan villages were chosen as the evaluation sites to comparatively analyze the indexes of intensive agriculture and schistosomiasis control effects. Results Compared with the demonstration area before construction, in 2014, the harden rates of ditches and village roads were increased by 49.57% and 39.33% respectively; and the proportion of agricultural machinery increased by 25%. The positive rate of serological tests of schistosomiasis was decreased by 81.74%. The Oncomelania hupensis snail area was decreased from 2.44 hm2 (2007) to 0 (2014). The awareness rate of schistosomiasis control knowledge and correct behavior rate of the residents were increased from 51.28% and 90.85% to 91.29% and 97.69% respectively. The experience of the demonstration area ensured the entire Guanghan District achieved the schistosomiasis transmission interruption criterion at the end of 2014. Conclusions The schistosomiasis control model of intensive agriculture combined with other comprehensive measures has a good effect on interrupting the endemic of schistosomiasis, and it can realize the sustainable development of the agricultural economy and schistosomiasis control.

The expression research of miR-210 in the elderly patients with COPD combined with ischemic stroke.

OBJECTIVE: We conduct this study to investigate the expression level of miR-210 in elderly patients with COPD combined with ischemic stroke and analyzed its application value as a sensitive diagnostic indicator. PATIENTS AND METHODS: 50 cases of elderly patients with COPD combined with ischemic stroke were selected as group A, 50 cases of elderly patients with COPD were selected as group B, 50 cases of elderly patients with ischemic stroke were selected as group C, and 50 cases of healthy volunteers as group D. Real-time PCR assay for quantification was used to detect the expression level of miR-210 in peripheral blood and receiver operating curve (ROC) was used to analyze the diagnostic sensitivity and accuracy. RESULTS: MiR-210 level of group A was the lowest, followed by group B and group C; group D had the highest levels. The difference was statistically significant (p<0.05). MiR-210 levels decreased with an increasing decline degree of lung function and the difference was statistically significant (p<0.05). After miR-210 diagnosis, COPD sensitivity was 85.6%, the specificity was 72.6%, accuracy (AUC) was 0.821, and 95% CI 0.632-0.924. CONCLUSIONS: The down-regulation of MiR-210 expression in the COPD combined with ischemic stroke can be regarded as a sensitive index in diagnosis of COPD and ischemic stroke.

Natural Endogenous Human Matriptase and Prostasin Undergo Zymogen Activation via Independent Mechanisms in an Uncoupled Manner.

The membrane-associated serine proteases matriptase and prostasin are believed to function in close partnership. Their zymogen activation has been reported to be tightly coupled, either as a matriptase-initiated proteolytic cascade or through a mutually dependent mechanism involving the formation of a reciprocal zymogen activation complex. Here we show that this putative relationship may not apply in the context of human matriptase and prostasin. First, the tightly coupled proteolytic cascade between matriptase and prostasin might not occur when modest matriptase activation is induced by sphingosine 1-phospahte in human mammary epithelial cells. Second, prostasin is not required and/or involved in matriptase autoactivation because matriptase can undergo zymogen activation in cells that do not endogenously express prostasin. Third, matriptase is not required for and/or involved in prostasin activation, since activated prostasin can be detected in cells expressing no endogenous matriptase. Finally, matriptase and prostasin both undergo zymogen activation through an apparently un-coupled mechanism in cells endogenously expressing both proteases, such as in Caco-2 cells. In these human enterocytes, matriptase is detected primarily in the zymogen form and prostasin predominantly as the activated form, either in complexes with protease inhibitors or as the free active form. The negligible levels of prostasin zymogen with high levels of matriptase zymogen suggests that the reciprocal zymogen activation complex is likely not the mechanism for matriptase zymogen activation. Furthermore, high level prostasin activation still occurs in Caco-2 variants with reduced or absent matriptase expression, indicating that matriptase is not required and/or involved in prostasin zymogen activation. Collectively, these data suggest that any functional relationship between natural endogenous human matriptase and prostasin does not occur at the level of zymogen activation.

[Prognostic significance of tumor deposits in patients with stage â ¢ colon cancer].

Objective: To investigate the prognostic significance of tumor deposits (TD) in patients with stage Ⅲ colorectal cancer who underwent radical resection. Methods: We retrospectively analyzed the clinicopathological data, and the impact of tumor deposits on the disease free survival (DFS) and overall survival (OS) in 206 patients with stage Ⅲ colorectal cancer using Kaplan-Meier and Cox regression analyses. Results: Among the 206 patients with stage Ⅲ colorectal cancer, there were 112 (54.4%) tumor deposit-positive (TD+ ) cases and 94 (45.6%) tumor deposit-negative (TD-) cases. The TD- and TD+ cases accounted for 10.6% and 21.4% of patients aged <50 years, 22.3% and 36.6% of patients with poorly differentiated carcinoma, and 16% and 29.5% of patients with neural invasion, respectively (P<0.05 for all). In addition, the neural invasion was more often found along with the increasing number of TD. However, multivariate analysis showed that TD was not significantly associated with DFS and OS of the patients. Among the patients with N1 stage cancer, the 3-year DFS rates of TD+ and TD- groups were 23.0% and 58.8%, and the median disease-free survival durations were 26 months and 43 months, respectively (P=0.002). Meanwhile, in the TD- group, the 3-year DFS rates in the patients with stage N1a~1b without tumor deposit and N1c cancer were 63.5% and 21.2%, and the median disease-free survival durations were 41 and 25 months, respectively (P=0.004). Conclusions: Tumor deposit is an independent unfavorable prognostic factor affecting the DFS. The number of tumor deposits may be an important factor affecting prognosis in stage Ⅲ colorectal cancer patients. Tumor deposits after radical resection are associated with neural invasion, and can be used as an independent factor of poor prognostic evaluation in patients with stage N1 colorectal cancer.

Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane.

Matriptase and prostasin, acting as a tightly coupled proteolytic cascade, were reported to be required for epidermal barrier formation in mouse skin. Here we show that, in human skin, matriptase and prostasin are expressed with an inverse pattern over the course of differentiation. Matriptase was detected primarily in epidermal basal keratinocytes and the basaloid cells in the outer root sheath of hair follicles and the sebaceous gland, where prostasin was not detected. In contrast, prostasin was detected primarily in differentiated cells in the epidermal granular layer, the inner root sheath of hair follicles, and the sebaceous gland, where matriptase expression is negligible. While co-expressed in the middle stage of differentiation, prostasin was detected as polarized patches, and matriptase at intercellular junctions. Targeting to different subcellular localizations is also observed in HaCaT human keratinocytes, in which matriptase was detected primarily at intercellular junctions, and prostasin primarily on membrane protrusion. Furthermore, upon induction of zymogen activation, free active prostasin remains cell-associated and free active matriptase is rapidly shed into the extracellular milieu. Our data suggest that matriptase and prostasin likely function as independent entities in human skin rather than as a tightly coupled proteolytic cascade as observed in mouse skin.

The high osteotomy cut of Dega procedure for developmental dysplasia of the hip in children under 6 years of age.

BACKGROUND: Dega osteotomy has gained popularity for the acetabular reconstruction of developmental dysplasia of the hip (DDH). A high-level Dega osteotomy combining the typical Dega procedure with an age limit of under 6 years and the higher pelvic cut height of the Salter osteotomy for walking DDH patients aged 6 or younger was advocated in our department. The purpose of this retrospective study is to introduce this modified Dega procedure and report the preliminary clinical and radiological results in DDH patients under 6. MATERIALS AND METHODS: From September 2000 to September 2010, a total of 162 patients with 191 dysplastic hips between 1.5 and 6 years old were managed with this high-level Dega osteotomy. From each patient's record, clinical status was retrospectively assessed using modified McKay criteria; radiological evaluations were assessed according to the modified Severin classification, the mean acetabular index (AI), Sharp angle and center-edge (CE) angle. Any occurrence of complications was recorded postoperatively. RESULTS: The average age at surgery was 3.6 years (range 1.5 to 6.0), mean duration of follow-up 11.3 years (range 5.0 to 16.9). The latest follow-up fineness (favorable and good) rate in clinical and radiological evaluations was 92.7 % and 91.1 % respectively. There was a marked trend toward normalization of radiographic indices during the follow-up period: the mean AI changed from 38.0° to 20.8°, the mean Sharp's angle decreased from 59.9° to 39.2°, and the mean CE angle increased from -10.7° to 29.4°, preoperatively and at the latest follow-up respectively. No major complications were recorded during follow-up. CONCLUSION: The high osteotomy cut Dega procedure for patients between 1.5 and 6 years of age was found to be sufficient for improving clinical and radiographic outcomes and inducing little morbidity and few complications for late-detected pediatric walking DDH patients.

Comparison of EGFR mutation rates in lung adenocarcinoma tissue and pleural effusion samples.

The goal of the current study was to investigate the differences in epidermal growth factor receptor (EGFR) mutation rates in tumor tissue and pleural effusion specimens from patients with lung adenocarcinoma. PCR amplification and gene sequencing were used to detect EGFR mutations in exons 18, 19, 20, and 21 in tumor tissue and pleural effusion samples from 50 patients with advanced lung adenocarcinoma. The EGFR mutation rate was 34.0% in tissue samples from patients with advanced lung adenocarcinoma. There were 11 cases with exon 19 mutations and 6 cases with exon 21 mutations. The EGFR mutation rate was 30.0% in pleural effusion specimens, including 10 cases with exon 19 mutation and 5 cases with exon 21 mutations. Although the tissue samples had a slightly higher mutation rate compared to the pleural effusion samples, the difference was not statistically significant. These results indicate that the EGFR mutation rate detected in pleural effusion specimens from patients with advanced lung adenocarcinoma is similar to that detected in tumor tissue samples. Therefore, pleural effusion specimens can potentially be used for EGFR mutation detection in advanced lung adenocarcinoma.

Matriptase Complexes and Prostasin Complexes with HAI-1 and HAI-2 in Human Milk: Significant Proteolysis in Lactation.

Significant proteolysis may occur during milk synthesis and secretion, as evidenced by the presence of protease-protease inhibitor complex containing the activated form of the type 2 transmembrane serine protease matriptase and the transmembrane Kunitz-type serine protease inhibitor HAI-1. In order to identify other proteolysis events that may occur during lactation, human milk was analyzed for species containing HAI-1 and HAI-2 which is closely related to HAI-1. In addition to the previously demonstrated matriptase-HAI-1 complex, HAI-1 was also detected in complex with prostasin, a glycosylphosphatidylinositol (GPI)-anchored serine protease. HAI-2 was also detected in complexes, the majority of which appear to be part of higher-order complexes, which do not bind to ionic exchange columns or immunoaffinity columns, suggesting that HAI-2 and its target proteases may be incorporated into special protein structures during lactation. The small proportion HAI-2 species that could be purified contain matriptase or prostasin. Human mammary epithelial cells are the likely cellular sources for these HAI-1 and HAI-2 complexes with matriptase and prostasin given that these protease-inhibitor complexes with the exception of prostasin-HAI-2 complex were detected in milk-derived mammary epithelial cells. The presence of these protease-inhibitor complexes in human milk provides in vivo evidence that the proteolytic activity of matriptase and prostasin are significantly elevated at least during lactation, and possibly contribute to the process of lactation, and that they are under tight control by HAI-1 and HAI-2.

N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.

The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.