Improving access to primary health care through financial innovation in rural China: a quasi-experimental synthetic difference-in-differences approach.

BACKGROUND: Inadequate financing constrains primary healthcare (PHC) capacity in many low- and middle-income countries, particularly in rural areas. This study evaluates an innovative PHC financing reform in rural China that aimed to improve access to healthcare services through supply-side integration and the establishment of a designated PHC fund. METHODS: We employed a quasi-experimental synthetic difference-in-differences (SDID) approach to analyze county-level panel data from Chongqing Province, China, spanning from 2009 to 2018. The study compared the impact of the reform on PHC access and per capita health expenditures in Pengshui County with 37 other control counties (districts). We assessed the reform's impact on two key outcomes: the share of outpatient visits at PHC facilities and per capita total PHC expenditure. RESULTS: The reform led to a significant increase in the share of outpatient visits at PHC facilities (14.92% points; 95% CI: 6.59-23.24) and an increase in per capita total PHC expenditure (87.30 CNY; 95% CI: 3.71-170.88) in Pengshui County compared to the synthetic control. These effects were robust across alternative model specifications and increased in magnitude over time, highlighting the effectiveness of the integrated financing model in enhancing PHC capacity and access in rural China. CONCLUSIONS: This research presents compelling evidence demonstrating that horizontal integration in PHC financing significantly improved utilization and resource allocation in rural primary care settings in China. This reform serves as a pivotal model for resource-limited environments, demonstrating how supply-side financing integration can bolster PHC and facilitate progress toward universal health coverage. The findings underscore the importance of sustainable financing mechanisms and the need for policy commitment to achieve equitable healthcare access.

Horizontal Integration and Financing Reform of Rural Primary Care in China: A Model for Low-Resource and Remote Settings.

Primary health care (PHC) systems are compromised by under-resourcing and inadequate governance, and fail to provide high-quality health care services in most low- and middle-income countries (LMICs). As a response to solve the problems of underfunding and understaffing, Pengshui County, an impoverished area in rural Chongqing, China, implemented a profound reform of its PHC delivery system in 2009, focusing on horizontal integration and financing mechanisms. This paper aims to present new evidence from the Pengshui model, and to assess the relevant changes over the past 10 years (2009-2018). An inductive approach was adopted, based on analysis of national and local policy documents and administrative data. From 2009 to 2018, the proportion of outpatients who sought first-contact care in rural community or township health centers increased from 29% (522,700 of 1,817,600) in 2009, to 40% (849,900 of 2,147,800) in 2018 (the national average in 2018 was 23%). Our findings suggest that many positive results have been achieved through the reform, and that innovations in financial governance and incentive mechanisms are the main driving forces behind the improvement. Pengshui County's experience has proven to be a successful experiment, particularly in rural and low-income areas.

Blending Donors with Different Molecular Weights: An Efficient Strategy to Resolve the Conflict between Coherence Length and Intermixed Phase in Polymer/Nonfullerene Solar Cells.

Long coherence lengths (CLs) of crystals and proper intermixed phase amount guarantee charge transport and exciton dissociate efficiently, which is crucial for organic solar cells (OSCs) to achieve high device performance. However, extending CLs usually reduces the intermixed phase, leading to an insufficient interface for exciton dissociation. Herein, a strategy using a binary polymer with different molecular weights as donor is employed, that is, poly(3-hexylthiophene-2,5-diyl) (P3HT) with high (P3HT-H) and low (P3HT-L) molecular weight are blended as donor, and (5Z,5'Z)-5,5'-(((4,4,9,9-tetraoctyl-4,9-dihydro-s-indaceno[1,2-b:5,6-b']dithiophene-2,7-diyl)bis(benzo[c][1,2,5]thiadiazole-7,4-diyl))bis(methanylylidene))bis(3-ethyl-2-thioxothiazolidin-4-one) (O-IDTBR) is used as acceptor. In kinetics, the entanglements of P3HT-H are relieved due to the higher molecular diffusivity of P3HT-L. In thermodynamics, the miscibility of P3HT-L/O-IDTBR, P3HT-H/O-IDTBR, and P3HT-L/P3HT-H blends increases in turn. Hence, P3HT forms a more ordered structure with longer CLs after adding P3HT-L, which also drives O-IDTBR dispersed in P3HT crystalline regions diffuse to the O-IDTBR crystalline regions to further self-organize. Consequently, the CLs of both P3HT and O-IDTBR are extended, while keeping the intermixed phase amount proper. The optimized microstructure boosts device performance from 7.03% to 7.80%, which is one of the highest values reported for P3HT/O-IDTBR blends. This is a novel way to solve the conflict mentioned above, which may provide guidance to finely regulating the morphology of the active layer.

Interaction between DUE-B and Treslin is required to load Cdc45 on chromatin in human cells.

A key step in the initiation of eukaryotic DNA replication is the binding of the activator protein Cdc45 to promote MCM helicase unwinding of the origin template. We show here that the c-myc origin DNA unwinding element-binding protein, DUE-B, interacts in HeLa cells with the replication initiation protein Treslin to allow Cdc45 loading onto chromatin. The chromatin loading of DUE-B and Treslin are mutually dependent, and the DUE-B-Treslin interaction is cell cycle-regulated to peak as cells exit G1 phase prior to the initiation of replication. The conserved C-terminal domain of DUE-B is required for its binding to TopBP1, Treslin, Cdc45, and the MCM2-7 complex, as well as for the efficient loading of Treslin, Cdc45, and TopBP1 on chromatin. These results suggest that DUE-B acts to identify origins by MCM binding and serves as a node for replication protein recruitment and Cdc45 transfer to the prereplication complex.

Protein phosphatase 2A and Cdc7 kinase regulate the DNA unwinding element-binding protein in replication initiation.

The DNA unwinding element (DUE)-binding protein (DUE-B) binds to replication origins coordinately with the minichromosome maintenance (MCM) helicase and the helicase activator Cdc45 in vivo, and loads Cdc45 onto chromatin in Xenopus egg extracts. Human DUE-B also retains the aminoacyl-tRNA proofreading function of its shorter orthologs in lower organisms. Here we report that phosphorylation of the DUE-B unstructured C-terminal domain unique to higher organisms regulates DUE-B intermolecular binding. Gel filtration analyses show that unphosphorylated DUE-B forms multiple high molecular weight (HMW) complexes. Several aminoacyl-tRNA synthetases and Mcm2-7 proteins were identified by mass spectrometry of the HMW complexes. Aminoacyl-tRNA synthetase binding is RNase A sensitive, whereas interaction with Mcm2-7 is nuclease resistant. Unphosphorylated DUE-B HMW complex formation is decreased by PP2A inhibition or direct DUE-B phosphorylation, and increased by inhibition of Cdc7. These results indicate that the state of DUE-B phosphorylation is maintained by the equilibrium between Cdc7-dependent phosphorylation and PP2A-dependent dephosphorylation, each previously shown to regulate replication initiation. Alanine mutation of the DUE-B C-terminal phosphorylation target sites increases MCM binding but blocks Cdc45 loading in vivo and inhibits cell division. In egg extracts alanine mutation of the DUE-B C-terminal phosphorylation sites blocks Cdc45 loading and inhibits DNA replication. The effects of DUE-B C-terminal phosphorylation reveal a novel S phase kinase regulatory mechanism for Cdc45 loading and MCM helicase activation.

Endocrine disruption screening by protein and gene expression of vitellogenin in freshly isolated and cryopreserved rainbow trout hepatocytes.

Xenobiotics may activate the estrogen receptor, resulting in alteration of normal endocrine functions in animals and humans. Consequently, this necessitates development of assay end points capable of identifying estrogenic xenobiotics. In the present study, we screened the potential estrogenicity of chemicals via their ability to induce vitellogenin (VTG) expression in cultured primary hepatocytes from male trout. A routine method for VTG detection measures the secretion of the protein by enzyme-linked immunosorbent assay (ELISA) in freshly isolated trout hepatocytes. However, this lengthy (6 days) culturing procedure requires that hepatocyte isolation is performed each time the assay is run. We optimized this methodology by investigating the utility of cryopreserved hepatocytes, shortening the incubation time, performing a quantitative real-time PCR (qPCR) method for VTG quantification, and verifying the model system with reference chemicals 17ß-estradiol, estrone, diethylstilbestrol, hexestrol, genistein, and a negative control, corticosterone. To test the performance of both freshly isolated and cryopreserved hepatocytes, mRNA was collected from hepatocytes following 24 h treatment for VTG gene expression analysis, whereas cell culture media was collected for a VTG ELISA 96 h post-treatment. EC50 values were obtained for each reference chemical except for corticosterone, which exhibited no induction of VTG gene or protein level. Our results show linear concordance between ELISA and qPCR detection methods. Although there was approximately 50% reduction in VTG inducibility following cryopreservation, linear concordance of EC50 values was found between freshly isolated and cryopreserved hepatocytes, indicating that cryopreservation does not alter the functional assessment of estrogen receptor activation and therefore VTG expression. These studies demonstrate that qPCR is a sensitive and specific method for detecting VTG gene expression that can be used together with cryopreserved trout hepatocytes for screening estrogenic chemicals, resulting in a reduction of the time required to perform the assay and enabling greater access to the model system through the approach of cryopreservation.

Preliminary study correlating CX3CL1/CX3CR1 expression with gastric carcinoma and gastric carcinoma perineural invasion.

AIM: To study the relationship between the CX3CL1 chemokine, its receptor CX3CR1, and gastric carcinoma/gastric carcinoma perineural invasion (PNI). METHODS: Thirty cases of gastric carcinoma were surgically resected (radical resection or palliative resection) between February 2012 and July 2012. Tumour and tumour-adjacent tissues were evaluated for the presence of CX3CL1 (ELISA) and CX3CR1 (immunohistochemistry and Western blotting) in an effort to analyse the relationship between CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma PNI. RESULTS: Of these 30 cases, 14 were PNI-positive (46.7%). No significant differences in CX3CL and CX3CR1 expression in tumour-adjacent tissues were found between the PNI positive and negative groups. Expression levels of CX3CL and CX3CR1 in tumour tissues were significantly higher than those in adjacent tissues (P < 0.01), and were significantly higher in tumour tissues from the PNI-positive group compared to the PNI-negative group (P < 0.01). CONCLUSION: CX3CL1/CX3CR1 expression may be associated with the occurrence and development of gastric carcinoma as well as gastric carcinoma PNI.

The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation.

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.

A critical role for IRAK4 kinase activity in Toll-like receptor-mediated innate immunity.

IRAK4 is a member of IL-1 receptor (IL-1R)-associated kinase (IRAK) family and has been shown to play an essential role in Toll-like receptor (TLR)-mediated signaling. We recently generated IRAK4 kinase-inactive knock-in mice to examine the role of kinase activity of IRAK4 in TLR-mediated signaling pathways. The IRAK4 kinase-inactive knock-in mice were completely resistant to lipopolysaccharide (LPS)- and CpG-induced shock, due to impaired TLR-mediated induction of proinflammatory cytokines and chemokines. Although inactivation of IRAK4 kinase activity did not affect the levels of TLR/IL-1R-mediated nuclear factor kappaB activation, a reduction of LPS-, R848-, and IL-1-mediated mRNA stability contributed to the reduced cytokine and chemokine production in bone marrow-derived macrophages from IRAK4 kinase-inactive knock-in mice. Both TLR7- and TLR9-mediated type I interferon production was abolished in plasmacytoid dendritic cells isolated from IRAK4 knock-in mice. In addition, influenza virus-induced production of interferons in plasmacytoid DCs was also dependent on IRAK4 kinase activity. Collectively, our results indicate that IRAK4 kinase activity plays a critical role in TLR-dependent immune responses.

The Toll-interleukin-1 receptor member SIGIRR regulates colonic epithelial homeostasis, inflammation, and tumorigenesis.

Despite constant contact with the large population of commensal bacteria, the colonic mucosa is normally hyporesponsive to these potentially proinflammatory signals. Here we report that the single immunoglobulin IL-1 receptor-related molecule (SIGIRR), a negative regulator for Toll-IL-1R signaling, plays a critical role in gut homeostasis, intestinal inflammation, and colitis-associated tumorigenesis by maintaining the microbial tolerance of the colonic epithelium. SIGIRR-deficient (Sigirr(-/-)) colonic epithelial cells displayed commensal bacteria-dependent homeostatic defects, as shown by constitutive upregulation of inflammatory genes, increased inflammatory responses to dextran sulfate sodium (DSS) challenge, and increased Azoxymethane (AOM)+DSS-induced colitis-associated tumorigenesis. Gut epithelium-specific expression of the SIGIRR transgene in the SIGIRR-deficient background reduced the cell survival of the SIGIRR-deficient colon epithelium, abrogated the hypersensitivity of the Sigirr(-/-) mice to DSS-induced colitis, and reduced AOM+DSS-induced tumorigenesis. Taken together, our results indicate that epithelium-derived SIGIRR is critical in controlling the homeostasis and innate immune responses of the colon to enteric microflora.

Interleukin-1 (IL-1)-induced TAK1-dependent Versus MEKK3-dependent NFkappaB activation pathways bifurcate at IL-1 receptor-associated kinase modification.

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is phosphorylated after it is recruited to the receptor, subsequently ubiquitinated, and eventually degraded upon IL-1 stimulation. Although a point mutation changing lysine 134 to arginine (K134R) in IRAK abolished IL-1-induced IRAK ubiquitination and degradation, mutations of serines and threonines adjacent to lysine 134 to alanines ((S/T)A (131-144)) reduced IL-1-induced IRAK phosphorylation and abolished IRAK ubiquitination. Through the study of these IRAK modification mutants, we uncovered two parallel IL-1-mediated signaling pathways for NFkappaB activation, TAK1-dependent and MEKK3-dependent, respectively. These two pathways bifurcate at the level of IRAK modification. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classical NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through IkappaBalpha phosphorylation and subsequent dissociation from NFkappaB but without IkappaBalpha degradation. These results provide significant insight to our further understanding of NFkappaB activation pathways.

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent.

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.

SIGIRR inhibits interleukin-1 receptor- and toll-like receptor 4-mediated signaling through different mechanisms.

The Toll-interleukin-1 receptor (TIR) domain-containing orphan receptor SIGIRR (single immunoglobulin interleukin-1 receptor-related protein) acts as a negative regulator of interleukin (IL)-1 and lipopolysaccharide (LPS) signaling. Endogenous SIGIRR transiently interacted with IL-1 receptor and the receptor-proximal signaling components (MyD88, IRAK, and tumor necrosis factor receptor-associated factor 6) upon IL-1 stimulation, indicating that SIGIRR interacts with the IL-1 receptor complex in a ligand-dependent manner. Similar interaction was also observed between SIGIRR and Toll-like receptor 4 receptor complex upon LPS stimulation. To identify the domains of SIGIRR required for its interaction with the Toll-like receptor 4 and IL-1 receptor complexes, several SIGIRR deletion mutants were generated, including DeltaN (lacking the extracellular immunoglobulin (Ig) domain with deletion of amino acids 1-119), DeltaC (lacking the C-terminal domain with deletion of amino acids 313-410), and DeltaTIR (lacking the TIR domain with deletion of amino acids 161-313). Whereas both the extracellular Ig domain and the intracellular TIR domains are important for SIGIRR to inhibit IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The extracellular Ig domain exerts its inhibitory role in IL-1 signaling by interfering with the heterodimerization of IL-1 receptor and IL-1RAcP, whereas the intracellular TIR domain inhibits both IL-1 and LPS signaling by attenuating the recruitment of receptor-proximal signaling components to the receptor. These results indicate that SIGIRR inhibits IL-1 and LPS signaling pathways through differential mechanisms.

Expression and purification of a novel mannose-binding lectin from Pinellia ternata.

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for "native" lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.

Molecular cloning and characterization of a novel mannose-binding lectin gene from Amorphophallus konjac.

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.

Molecular cloning and characterization of a novel lectin gene from Zephyranthes candida.

A new lectin gene was cloned from Zephyranthes candida by using RACE-PCR. The full-length cDNA of Zephyranthes candida agglutinin (ZCA) was 647 bp and contained a 477 bp open reading frame encoding a 159 amino acid protein. Zephyranthes candida lectin gene was found to encode a precursor lectin with signal peptide and had extensive homology with those of other plant lectins. Molecular modeling of ZCA indicated that the three-dimensional structure of ZCA strongly resembles that of the snowdrop lectin, implying ZCA may have the similar insecticidal functions with GNA.

Isolation and expression of cold-regulated cDNA from Chinese cabbage (Brassica pekinensis).

It has been established that changes in gene expression occur during cold acclimation in a wide range of plants. Here we present a novel cDNA encoding a protein with a calculated molecular mass of 25 kDa, designated cor25, from Brassica pekinensis cv. Zaoshu 5 (Chinese cabbage) that was similar with a cold-regulated Arabidopsis thaliana L. gene, cor47. Protein sequence alignment showed that COR25 had 53.5% of identity with COR47 and 59.6% of identity with ERD10, an Arabidopsis thaliana L. early-responsive gene to dehydration stress. Northern blot analysis revealed that the cor25 expression was cold inducible. Moreover, the transcripts of the cor25 accumulated in plants in response to exogenous application of ABA and water stress. The possible functions of the cold-regulated gene and the mechanism for plants to cope with low temperature are discussed.

Molecular cloning and characterization of a novel lectin gene from Lycoris radiata.

By the use of RACE-PCR with RNA extracted from Lycoris radiata young leaves, the full-length cDNA of L. radiata agglutinin (LRA) was cloned. The full-length cDNA of LRA was 669 bp and contained a 477 bp open reading frame encoding a 158 amino acid protein. Through comparative analysis of lra gene and its deduced amino acid sequence with those of other Amaryllidaceae species, it was found that lra encoded a precursor lectin with signal peptide. LRA was a mannose-binding lectin with three mannose-binding boxes like lectins from other Amaryllidaceae species. Southern blot analysis of genomic DNA revealed that lra belonged to a low copy gene family. Northern blot analysis demonstrated that lra constitutively expressed in various plant tissues including leaf, root, stem and inflorescence, and highly expressed in the inflorescence.