Intratumoral Microbiota: Unraveling their Oncogenic Impact on Cancer Progression With Focus on Breast Cancer Therapeutic Outcomes.

The gut microbiota has been implicated in many cancers through the secretion of blood-traveling metabolites or activation of oncogenic signaling. Currently, specific microbial signatures have been detected in the human breast, which are different from other microbial-rich compartments, such as the intestine and skin. Changes in the breast microbiome profile have been shown to positively or negatively correlate with breast cancer development, progression, and therapeutic outcomes. However, studies regarding the role and underlying mechanism of intratumoral microbiota in breast cancer have remained concealed. This review aimed to provide an overview of the role of the intratumoral microbiome in tumorigenesis and tumor progression, and how these intratumoral microbiota affect breast cancer. We also discuss the potential of using the intratumoral microbiome as a biomarker or treatment alternative in breast cancers.

The Use of Schisandrin B to Combat Triple-Negative Breast Cancers by Inhibiting NLRP3-Induced Interleukin-1ß Production.

Triple-negative breast cancer (TNBC) is the most aggressive and fatal breast cancer subtype. Nowadays, chemotherapy remains the standard treatment of TNBC, and immunotherapy has emerged as an important alternative. However, the high rate of TNBC recurrence suggests that new treatment is desperately needed. Schisandrin B (Sch B) has recently revealed its anti-tumor effects in cancers such as cholangiocarcinoma, hepatoma, glioma, and multi-drug-resistant breast cancer. However, there is still a need to investigate using Sch B in TNBC treatment. Interleukin (IL)-1ß, an inflammatory cytokine that can be expressed and produced by the cancer cell itself, has been suggested to promote BC proliferation and progression. In the current study, we present evidence that Sch B can significantly suppress the growth, migration, and invasion of TNBC cell lines and patient-derived TNBC cells. Through inhibition of inflammasome activation, Sch B inhibits interleukin (IL)-1ß production of TNBC cells, hindering its progression. This was confirmed using an NLRP3 inhibitor, OLT1177, which revealed a similar beneficial effect in combating TNBC progression. Sch B treatment also inhibits IL-1ß-induced EMT expression of TNBC cells, which may contribute to the anti-tumor response.

Long-Term Usage of Proton Pump Inhibitors Associated with Prognosis in Patients with Colorectal Cancer.

The dose-response effect of proton pump inhibitors on colorectal cancer prognosis is still under exploration. This population-based study in Taiwan was designed to examine the effect of proton pump inhibitors on overall death, colorectal cancer-specific death, and recurrence in colorectal cancer patients with different cumulative proton pump inhibitor dose levels. This cohort study was based on the Taiwan Cancer Registry and Taiwan National Health Insurance Research Database from 2005 to 2020. After frequency matching with a 1:1 ratio, a total of 20,889 users with proton pump inhibitors and 20,889 without proton pump inhibitors were analyzed. The cumulative defined daily dose level of proton pump inhibitor was stratified to explore the dose-response relationship. A proton pump inhibitor exposure cumulative defined daily dose > 60 after colorectal cancer diagnosis had higher risk of all-cause death than non-proton pump inhibitor users with adjusted hazard ratios of 1.10 (95% CIs: 1.04-1.18). For recurrence, a proton pump inhibitor exposure cumulative defined daily dose > 60 had reduced recurrence risk with an adjusted hazard ratio of 0.84 (95% CIs: 0.76-0.93). This study demonstrated that the long-term use of proton pump inhibitors in patients with colorectal cancer was associated with an increased risk of death that related to the proton pump inhibitor exposure cumulative defined daily dose > 60 and had different dose-response effect in various dose level.

Mechanism of CDK4/6 Inhibitor Resistance in Hormone Receptor-positive Breast Cancer and Alternative Treatment Strategies.

Breast cancer (BC) is a common malignancy in women, with hormone receptor (HR)-positive subtype responsible for approximately 70% of cases. Currently, patients with metastatic HR-positive BC rely on endocrine therapy and cyclin-dependent kinase (CDK)-4/6 inhibitors for treatment. Currently, approved CDK4/6 inhibitors include palbociclib, ribociclib, and abemaciclib. However, clinical evidence of CDK-4/6 inhibitor resistance is emerging, suggesting that the gap in the knowledge of its resistance mechanism requires further investigation. This review discusses the mechanisms of CDK4/6 inhibitor resistance in BC, including both intrinsic and extrinsic mechanisms. We also discuss possible alternative strategies to overcome CDK4/6 inhibitor resistance in future clinical applications.

Metaplastic Carcinoma of the Axillary Breast With Heterologous Mesenchymal (Chondroid) Differentiation: A Difficult Case and Literature Review.

Metaplastic breast carcinoma is an invasive carcinoma with a high differentiation rate of the neoplastic epithelium toward mesenchymal-like epithelium. It comprises of only less than 1% of all breast cancers. Although 80% to 90% of metaplastic breast carcinomas are triple-negative cancers, they usually have worse outcomes than other triple-negative breast cancers (TNBCs). Metaplastic carcinoma is also often refractory to cytotoxic chemotherapy. Here, we reported a case of a 61-year-old female patient, presenting with a solitary and pedunculated mass in the right axillary tail breast tissue, whose biopsy revealed metaplastic breast carcinoma with chondroid differentiation. She had failed neoadjuvant chemotherapy and immunotherapy. Although she received debulking surgery, the tumor regrew even faster before surgery. Despite receiving palliative chemotherapy, the patient died 11 weeks after surgery. This case draws attention to physicians that early recognition and surgery may be more beneficial than chemotherapy in combating metaplastic breast carcinoma.

Therapeutic Peptide RF16 Derived from CXCL8 Inhibits MDA-MB-231 Cell Invasion and Metastasis.

Interleukin (IL)-8 plays a vital role in regulating inflammation and breast cancer formation by activating CXCR1/2. We previously designed an antagonist peptide, (RF16), to inhibits the activation of downstream signaling pathways by competing with IL-8 in binding to CXCR1/2, thereby inhibiting IL-8-induced chemoattractant monocyte binding. To evaluate the effect of the RF16 peptide on breast cancer progression, triple-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells were used to investigate whether RF16 can inhibit the IL-8-induced breast cancer metastasis. Using growth, proliferation, and invasiveness assays, the results revealed that RF16 reduced cell proliferation, migration, and invasiveness in MDA-MB-231 cells. The RF16 peptide also regulated the protein and mRNA expressions of epithelial-mesenchymal transition (EMT) markers in IL-8-stimulated MDA-MB-231 cells. It also inhibited downstream IL-8 signaling and the IL-8-induced inflammatory response via the mitogen-activated protein kinase (MAPK) and Phosphoinositide 3-kinase (PI3K) pathways. In the xenograft tumor mouse model, RF16 synergistically reinforces the antitumor efficacy of docetaxel by improving mouse survival and retarding tumor growth. Our results indicate that RF16 significantly inhibited IL-8-stimulated cell growth, migration, and invasion in MDA-MB-231 breast cancer cells by blocking the activation of p38 and AKT cascades. It indicated that the RF16 peptide may serve as a new supplementary drug for breast cancer.

Biomechanical evaluation of an osteoporotic anatomical 3D printed posterior lumbar interbody fusion cage with internal lattice design based on weighted topology optimization.

In this study, we designed and manufactured a posterior lumbar interbody fusion cage for osteoporosis patients using 3D-printing. The cage structure conforms to the anatomical endplate's curved surface for stress transmission and internal lattice design for bone growth. Finite element (FE) analysis and weight topology optimization under different lumbar spine activity ratios were integrated to design the curved surface (CS-type) cage using the endplate surface morphology statistical results from the osteoporosis patients. The CS-type and plate (P-type) cage biomechanical behaviors under different daily activities were compared by performing non-linear FE analysis. A gyroid lattice with 0.25 spiral wall thickness was then designed in the internal cavity of the CS-type cage. The CS-cage was manufactured using metal 3D printing to conduct in vitro biomechanical tests. The FE analysis result showed that the maximum stress values at the inferior L3 and superior L4 endplates under all daily activities for the P-type cage implantation model were all higher than those for the CS-type cage. Fracture might occur in the P-type cage because the maximum stresses found in the endplates exceeded its ultimate strength (about 10 MPa) under flexion, torsion and bending loads. The yield load and stiffness of our designed CS-type cage fall into the optional acceptance criteria for the ISO 23089 standard under all load conditions. This study approved a posterior lumbar interbody fusion cage designed to have osteoporosis anatomical curved surface with internal lattice that can achieve appropriate structural strength, better stress transmission between the endplate and cage, and biomechanically tested strength that meets the standard requirements for marketed cages.

Development and Biomechanical Evaluation of an Anatomical 3D Printing Modularized Proximal Inter-Phalangeal Joint Implant Based on the Computed Tomography Image Reconstructions.

In this study, we developed a modularized proximal interphalangeal (PIP) joint implant that closely resembles the anatomical bone articular surface and cavity contour based on computed tomography (CT) image reconstruction. Clouds of points of 48 groups reconstructed phalanx articular surfaces of CT images, including the index, middle, ring, and little fingers, were obtained and fitted to obtain the articular surface using iterative closest points algorithm. Elliptical-cone stems, including the length, the major and minor axis at the stem metaphyseal/diaphyseal side for the proximal and middle phalanxes, were designed. The resurfacing PIP joint implant components included the bi-condylar surface for the proximal phalanx with elliptical-cone stem, ultra-high molecular weight polyethylene bi-concave articular surface for middle phalanx with hook mechanism, and the middle phalanx with elliptical-cone stem. Nine sets of modularized designs were made to meet the needs of clinical requirements and the weakness structure from the nine sets, that is, the worst structure case combination was defined and manufactured using titanium alloy three-dimensional (3D) printing. Biomechanical tests including anti-loosening pull-out strength for the proximal phalanx, elliptical-cone stem, and articular surface connection strength for the middle phalanx, and static/dynamic (25000 cycles) dislocation tests under three daily activity loads for the PIP joint implant were performed to evaluate the stability and anti-dislocation capability. Our experimental results showed that the pull-out force for the proximal phalanx implant was 727.8N. The connection force for the hook mechanism to cone stem of the middle phalanx was 49.9N and the hook mechanism was broken instead of stem pull out from the middle phalanx. The static dislocation forces/dynamic fatigue limits (pass 25000 cyclic load) of daily activities for piano-playing, pen-writing, and can-opening were 525.3N/262.5N, 316.0N/158N, and 115.0N/92N, respectively, and were higher than general corresponding acceptable forces of 19N, 17N, and 45N from the literatures. In conclusion, our developed modularized PIP joint implant with anatomical articular surface and elliptical-cone stem manufactured by titanium alloy 3D printing could provide enough joint stability and the ability to prevent dislocation.

Short-term and long-term outcomes of free flap reconstruction versus amputation for diabetic foot reconstruction in patients with end-stage renal disease.

BACKGROUND: In Taiwan, the prevalence of diabetes mellitus complicated by end-stage renal disease (ESRD) has been increasing and diabetes-related foot amputation is commonplace. In recent years, limb salvage has become top priority. The long-term outcomes of patients on hemodialysis undergoing diabetic foot reconstruction using free flaps remain unknown. METHODS: Data from the National Health Insurance Research Database on hemodialysis patients with type 2 diabetes who received amputation or free flap reconstruction surgery for diabetic foot ulcer were analyzed from 2000 to 2013 using International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) codes. After 1:4 propensity score matching, 86 and 344 patients were assigned to the free flap reconstruction and amputation groups, respectively. RESULTS: The 5-year survival rate was significantly higher in patients who received free flap compared to the amputated group (1-year survival rate = 80.0% vs. 67.6%, p = 0.030; 3-year survival rate = 49.7% vs. 35.5%, p = 0.024; 5-year rate=30.1% vs. 19.9%, p = 0.018; however, after 5 years, the overall long-term survival rate was similar in both groups (p = 0.064). Patients who had lower limb amputation after flap reconstruction were susceptible to mortality (adjusted HR = 1.39; p = 0.069). Peripheral arterial disease was a dependent risk factor (HR = 1.45; p = 0.037) for long-term survival, whereas old age (> 75 years; HR = 1.65; p = 0.004), cerebrovascular disease (adjusted HR = 1.36; p = 0.011), and sepsis (adjusted HR = 1.85; p = 0.035) served as independent risk factors. Hemodialysis patients with diabetic foot ulcer who had limb salvaged showed a higher 5-year survival rate as compared to the amputated group.

Design Criteria for Patient-specific Mandibular Continuity Defect Reconstructed Implant with Lightweight Structure using Weighted Topology Optimization and Validated with Biomechanical Fatigue Testing.

This study developed design criterion for patient-specific reconstructed implants with appearance consideration and structural optimization of various mandibular continuity defects. The different mandible continuity defects include C (from left to right canines), B (from 1st premolar to 3rd molar), and A (from 3rd molar to ramus) segments defined based on the mandible image. The finite element (FE) analysis and weighted topology optimization methods were combined to design internal support beam structures within different reconstructed implants with corresponding occlusal conditions. Five continuity mandibular defects (single B/C/A+B and combination of B+C and B+C+B segments) were restored using additive manufacturing (AM) reconstructed implant and bone plate to confirm reasonable design criterion through biomechanical fatigue testing. The worst mandible strength was filtered based on the material mechanics and results from segmental bone length, thickness, and height statistics from the established database containing mandible images of 105 patients. The weighted optimization analysis results indicated that the sizes and positions of internal supporting beams within the reconstructed C, B, and A+B implants can be defined parametrically through corresponding segmental bone length, width, and height. The FE analysis found that the weight variation percentage between the parametric designed implants and original core solid implants in the C, B, and A+B was reduced by 54.3%, 63.7%, and 69.7%, respectively. The maximum stress values of the reconstructed implant and the remaining bone were not obviously reduced but the stress values were far lower than the material ultimate strength. The biomechanical fatigue testing indicated that all cases using the AM reconstructed implant could pass the 250,000 dynamic load. However, condyle head, bone plate fracture, and bone screw loosening could be found in cases using bone plates. This study developed a design criterion for patient-specific reconstructed implants for various mandibular continuity defects applicable for AM to further clinical use.

Development of a method for dansylation of metabolites using organic solvent-compatible buffer systems for amine/phenol submetabolome analysis.

Metabolomics, which serves as a readout of biological processes and diseases monitoring, is an informative research area for disease biomarker discovery and systems biology studies. In particular, reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) has become a powerful and popular tool for metabolomics analysis, enabling the detection of most metabolites. Very polar and ionic metabolites, however, are less easily detected because of their poor retention in RP columns. Dansylation of metabolites simplifies the sub-metabolome analysis by reducing its complexity and increasing both hydrophobicity and ionization ability. However, the various metabolite concentrations in clinical samples have a wide dynamic range with highly individual variation in total metabolite amount, such as in saliva. The bicarbonate buffer typically used in dansylation labeling reactions induces solvent stratification, resulting in poor reproducibility, selective sample loss and an increase in false-determined metabolite peaks. In this study, we optimized the dansylation protocol for samples with wide concentration range of metabolites, utilizing diisopropylethylamine (DIPEA) or tri-ethylamine (TEA) in place of bicarbonate buffer, and presented the results of a systemic investigation of the influences of individual processes involved on the overall performance of the protocol. In addition to achieving high reproducibility, substitution of DIPEA or TEA buffer resulted in similar labeling efficiency of most metabolites and more efficient labeling of some metabolites with a higher pKa. With this improvement, compounds that are only present in samples in trace amounts can be detected, and more comprehensive metabolomics profiles can be acquired for biomarker discovery or pathway analysis, making it possible to analyze clinical samples with limited amounts of metabolites.

Is Colectomy Associated with the Risk of Type 2 Diabetes in Patients without Colorectal Cancer? A Population-Based Cohort Study.

Type 2 diabetes might be influenced by colonic disease; however, the association between colonic resection and type 2 diabetes has rarely been discussed. This population-based cohort study explored the association between colectomy and type 2 diabetes in patients without colorectal cancer. A total of 642 patients who underwent colectomy for noncancerous diseases at any time between 2000 and 2012 in the National Health Insurance Research Database of Taiwan were enrolled. The enrolled patients were matched with 2568 patients without colectomy at a 1:4 ratio using a propensity score that covered age, sex, and comorbidities. The risk of type 2 diabetes was assessed using a Cox proportional hazards model. The mean (standard deviation) follow-up durations in colectomy cases and non-colectomy controls were 4.9 (4.0) and 5.6 (3.6) years, respectively; 65 (10.1%) colectomy cases and 342 (15.5%) non-colectomy controls developed type 2 diabetes. After adjustment, colectomy cases still exhibited a decreased risk of type 2 diabetes (adjusted HR = 0.80, 95% CI: 0.61-1.04). A stratified analysis for colectomy type indicated that patients who underwent right or transverse colectomy had a significantly lower risk of developing type 2 diabetes (adjusted HR = 0.57, 95% CI: 0.34-0.98). In the present study, colectomy tended to be at a reduced risk of type 2 diabetes in patients without colorectal cancer, and right or transverse colectomies were especially associated with a significantly reduced risk of type 2 diabetes.

Interleukin-10: A double-edged sword in breast cancer.

Breast cancer (BC) is a frequently diagnosed cancer among women worldwide. Currently, BC can be divided into different subgroups according to the presence of the following hormone receptors: estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Each of these subgroups has different treatment strategies. However, the presence of new metastatic lesions and patient deterioration suggest resistance to a given treatment. Various lines of evidence had shown that cytokines are one of the important mediators of tumor growth, invasion, metastasis, and treatment resistance. Interleukin-10 (IL-10) is an immunoregulatory cytokine, and acts as a poor prognostic marker in many cancers. The anti-inflammatory IL-10 blocks certain effects of inflammatory cytokines. It also antagonizes the co-stimulatory molecules on the antigen-presenting cells. Here, we review the current knowledge on the function and molecular mechanism of IL-10, and recent findings on how IL-10 contributes to the progression of BC.

Geometric accuracy of an acrylonitrile butadiene styrene canine tibia model fabricated using fused deposition modelling and the effects of hydrogen peroxide gas plasma sterilisation.

BACKGROUND: Three-dimensional (3D) printing techniques have been used to produce anatomical models and surgical guiding instruments in orthopaedic surgery. The geometric accuracy of the 3D printed replica may affect surgical planning. This study assessed the geometric accuracy of an acrylonitrile butadiene styrene (ABS) canine tibia model printed using fused deposition modelling (FDM) and evaluated its morphological change after hydrogen peroxide (H2O2) gas plasma sterilisation. The tibias of six canine cadavers underwent computed tomography for 3D reconstruction. Tibia models were fabricated from ABS on a 3D printer through FDM. Reverse-engineering technology was used to compare morphological errors (root mean square; RMS) between the 3D-FDM models and virtual models segmented from original tibia images (3D-CT) and between the models sterilised with H2O2 gas plasma (3D-GAS) and 3D-FDM models on tibia surface and in cross-sections at: 5, 15, 25, 50, 75, 85, and 95% of the tibia length. RESULTS: The RMS mean ± standard deviation and average positive and negative deviation values for all specimens in EFDM-CT (3D-FDM vs. 3D-CT) were significantly higher than those in EGAS-FDM (3D-GAS vs. 3D-FDM; P < 0.0001). Mean RMS values for EFDM-CT at 5% bone length (proximal tibia) were significantly higher than those at the other six cross-sections (P < 0.0001). Mean RMS differences for EGAS-FDM at all seven cross-sections were nonsignificant. CONCLUSIONS: The tibia models fabricated on an FDM printer had high geometric accuracy with a low RMS value. The surface deviation in EFDM-CT indicated that larger errors occurred during manufacturing than during sterilisation. Therefore, the model may be used for surgical rehearsal and further clinically relevant applications in bone surgery.

Minimally Invasive Surgery Is Feasible in Patients with Liver and Kidney Transplantation.

BACKGROUND Minimally invasive surgery (MIS) has rapidly advanced, but its use in transplant patients has lagged. We share our experience of MIS for patients after kidney and liver transplantation and compare our results with similar studies in the literature. MATERIAL AND METHODS This study included 14 MIS (12 laparoscopic, 2 transvaginal) procedures for 13 transplant cases (6 liver and 7 kidney) done from May 2006 to May 2018. Gastrointestinal surgery was performed in 6 cases: appendectomy performed 8 months after liver transplant and 16 months after kidney transplant in 2 cases, radical right hemi-colectomy performed 6 weeks after liver transplant in 1 case; exploration for chylous ascites 6 months after liver transplant in 1 case, sleeve gastrectomy performed 3 years after kidney transplant in 1 case, and partial hepatectomy performed 12 years after kidney transplant in 1 case. For urological problems, 2 patients received ipsilateral right-side nephroureterectomy performed 10 and 12 years after kidney transplant, and 1 patient received contralateral left-side nephroureterectomy performed 12 years after kidney transplant. The 2 liver transplant patients with huge incisional hernias received repair approximately 3 and 2 years after liver transplant. Three patients underwent gynecological surgery: 2 transvaginal for pelvic floor reconstruction in 1 patient with liver transplant and 1 hysterectomy in a kidney transplant patient, and 1 laparoscopic-assisted hysterectomy in a kidney transplant patient. We retrospectively analyzed the clinical presentation, operative findings, operation time, postoperative complications, and length of stay. RESULTS The postoperative course was uneventful, with early resumption of oral intake, including immunosuppressants administered the same as in the non-transplant patients. All surgical procedures in these transplant patients were achieved without conversion, showed stable kidney and liver function, had better surgical outcomes in comparison with traditional surgery, and most of them were discharged within 1 week. CONCLUSIONS Laparoscopic and non-laparoscopic MIS surgery are feasible and safe for abdominal organ transplant patients and are helpful for timely intervention in cases with acute abdomen. No adjustment of immunosuppressant is usually needed, as oral intake can be resumed very soon after surgery.

The impact of colectomy on the risk of cardiovascular disease among patients without colorectal cancer.

Cardiometabolic disorders were discussed and might be changed by microbiota in recent years. Since the colon acts as the primary reservoir of microbiota, we designed the present study to explore the association between colectomy and cardiovascular disease (CVD). We identified a total of 18,424 patients who underwent colectomy between 2000-2012 for reasons other than colorectal cancer from the National Health Insurance Research Database of Taiwan. Patients were matched with 18,424 patients without colectomy using a 1:1 propensity score by age, sex, and comorbidity. Cox proportional-hazards regression was used to assess the risk of CVD. Patients with colectomy were found to be at lower risk of CVD (hazard ratio [HR]: 0.95, 95% confidence interval [CI] = 0.90-0.99) than patients without colectomy. Stratified analysis according to the type of surgery revealed patients who underwent cecectomy and right hemicolectomy were at lower risk of CVD (cecectomy: adjusted HR [aHR] = 0.77, 95% CI = 0.64-0.94; right hemicolectomy: aHR = 0.88, 95% CI = 0.82-0.96). Patients who underwent left hemicolectomy were at higher risk of CVD (aHR = 1.19, 95% CI = 1.08-1.32). Our results indicate that the different colectomy procedures influence the risk for the CVD differently.

Electronic structures of 24-valence-electron full Heusler compounds investigated by density functional and GW calculations.

The electronic structures of Fe-based and Ru-based full Heusler compounds have been investigated systematically by density functional theory (DFT) with PBE, PBE + U, and HSE06 exchange-correlation (XC) functionals. In order to have a better systematic and quantitative comparison between the results of different approximations, the average deviation of eigenvalues (ADE) between any two electronic band structures were calculated. From quantitative analysis of the ADEs, we have shown that different XC functionals used in the DFT calculations will result in very different and inconsistent electronic band structures. However, the discrepancies are dramatically reduced and get more consistent band structures after the GW calculations. Furthermore, comparing the experimental and calculated Seebeck coefficients and band-gap values of Fe2VAl, it implies that the GW methods including dynamically screened Coulomb interactions are more reliable than DFT with PBE or HSE06 functionals. Conclusively, contrast to the fact that DFT methods give inconsistent band structures when using different XC functionals, the GW methods have better predictive power for the band structures of Fe-based and Ru-based full Heusler compounds.

Facilitating chemical and biochemical experiments with electronic microcontrollers and single-board computers.

Since the advent of modern science, researchers have had to rely on their technical skills or the support of specialized workshops to construct analytical instruments. The notion of the 'fourth industrial revolution' promotes construction of customized systems by individuals using widely available, inexpensive electronic modules. This protocol shows how chemists and biochemists can utilize a broad range of microcontroller boards (MCBs) and single-board computers (SBCs) to improve experimental designs and address scientific questions. We provide seven example procedures for laboratory routines that can be expedited by implementing this technology: (i) injection of microliter-volume liquid plugs into microscale capillaries for low-volume assays; (ii) transfer of liquid extract to a mass spectrometer; (iii) liquid-gas extraction of volatile organic compounds (called 'fizzy extraction'), followed by mass spectrometric detection; (iv) monitoring of experimental conditions over the Internet cloud in real time; (v) transfer of analytes to a mass spectrometer via a liquid microjunction interface, data acquisition, and data deposition into the Internet cloud; (vi) feedback control of a biochemical reaction; and (vii) optimization of sample flow rate in direct-infusion mass spectrometry. The protocol constitutes a primer for chemists and biochemists who would like to take advantage of MCBs and SBCs in daily experimentation. It is assumed that the readers have not attended any courses related to electronics or programming. Using the instructions provided in this protocol and the cited material, readers should be able to assemble simple systems to facilitate various procedures performed in chemical and biochemical laboratories in 1-2 d.

Risk of breast cancer in women with non-lactational mastitis.

Little is known regarding the association of non-lactational mastitis women with breast cancer risk. This population-based cohort study examined the breast cancer risk in women with non-lactational mastitis. We identified 3,091 women with non-lactational mastitis between 2000 and 2011 using the Taiwan National Health Insurance Research Database. We performed 1:4 propensity score matching by age, socioeconomic status and comorbidities and identified 12,364 women without non-lactational mastitis. The mean age of women with non-lactational mastitis was 37.9 years; these women had a higher breast cancer risk than the comparison group (adjusted hazard ratio = 1.94, 95% confidence interval: 1.30-2.90). The incidence rates of breast cancer in women with non-lactational mastitis and the comparison group were 14.79 and 7.57 per 10,000 person-years, respectively. Furthermore, non-lactational mastitis was a risk factor for breast cancer in women aged <50 years, women with lower socioeconomic status and women with hormonal medication (p < 0.05). Women who had more episodes of non-lactational mastitis had a higher risk of developing a breast cancer. Thus, the risk of breast cancer in women with non-lactational mastitis is significantly higher than those without non-lactational mastitis.

Temporal Analysis of Conformers in the Course of pH Scan Directed by Urea-Urease Reaction-A "Protein Clock".

We demonstrate an analytical approach to monitor the effect of pH on protein tertiary structure. An autocatalytic enzyme reaction is used to stimulate refolding of proteins during real-time analysis. The method takes advantage of a nonlinear pH ramp generated by the urea-urease clock reaction. In this study, alterations to the structures of model proteins were monitored by mass spectrometry (charge pattern shift) and fluorometry (tryptophan fluorescence quenching). The pH measurements were conducted at different points of the sample flow line by different methods to minimize artifacts. Interestingly, different protein ions (corresponding to native and unfolded proteins) show distinct temporal mass spectral profiles, which reveal gradual refolding and concomitant deprotonation of higher charge state ions in the course of the clock reaction. Every multiply charged ion of a protein is characterized with its own "clock time". This approach does not require major modification of standard instrumentation. It enables determination of "high sensitivity" pH intervals for small and large molecules within a single experiment. Thus, it can be useful for characterizing the protein folding in response to pH change.