Application of Bayesian phylogenetic inference modelling for evolutionary genetic analysis and dynamic changes in 2019-nCoV.

The novel coronavirus (2019-nCoV) has recently caused a large-scale outbreak of viral pneumonia both in China and worldwide. In this study, we obtained the entire genome sequence of 777 new coronavirus strains as of 29 February 2020 from a public gene bank. Bioinformatics analysis of these strains indicated that the mutation rate of these new coronaviruses is not high at present, similar to the mutation rate of the severe acute respiratory syndrome (SARS) virus. The similarities of 2019-nCoV and SARS virus suggested that the S and ORF6 proteins shared a low similarity, while the E protein shared the higher similarity. The 2019-nCoV sequence has similar potential phosphorylation sites and glycosylation sites on the surface protein and the ORF1ab polyprotein as the SARS virus; however, there are differences in potential modification sites between the Chinese strain and some American strains. At the same time, we proposed two possible recombination sites for 2019-nCoV. Based on the results of the skyline, we speculate that the activity of the gene population of 2019-nCoV may be before the end of 2019. As the scope of the 2019-nCoV infection further expands, it may produce different adaptive evolutions due to different environments. Finally, evolutionary genetic analysis can be a useful resource for studying the spread and virulence of 2019-nCoV, which are essential aspects of preventive and precise medicine.

A systems-level approach for investigating organophosphorus pesticide toxicity.

The full understanding of the single and joint toxicity of a variety of organophosphorus (OP) pesticides is still unavailable, because of the extreme complex mechanism of action. This study established a systems-level approach based on systems toxicology to investigate OP pesticide toxicity by incorporating ADME/T properties, protein prediction, and network and pathway analysis. The results showed that most OP pesticides are highly toxic according to the ADME/T parameters, and can interact with significant receptor proteins to cooperatively lead to various diseases by the established OP pesticide -protein and protein-disease networks. Furthermore, the studies that multiple OP pesticides potentially act on the same receptor proteins and/or the functionally diverse proteins explained that multiple OP pesticides could mutually enhance toxicological synergy or additive on a molecular/systematic level. To the end, the integrated pathways revealed the mechanism of toxicity of the interaction of OP pesticides and elucidated the pathogenesis induced by OP pesticides. This study demonstrates a systems-level approach for investigating OP pesticide toxicity that can be further applied to risk assessments of various toxins, which is of significant interest to food security and environmental protection.

Treatment and outcomes of tumor-induced osteomalacia associated with phosphaturic mesenchymal tumors: retrospective review of 12 patients.

BACKGROUND: Tumor-induced osteomalacia (TIO) is a rare paraneoplastic syndrome characterized by severe hypophosphatemia and osteomalacia. Nonspecific symptoms make the diagnosis elusive. In addition, locating the responsible tumor(s) is challenging. The aim of this study was to investigate the clinical management and outcomes of TIO. METHODS: The clinical features, diagnostic procedures, treatment, and outcomes of 12 patients were reviewed retrospectively. RESULTS: The cohort comprised six men and six women (mean age 45.5 ± 9.9 years, range 23-61 years). The mean duration of disease was 3.7 ± 2.6 years. All patients manifested progressive bone pain, muscle weakness, and/or difficulty walking. Serum phosphorus concentrations were low in all patients (mean 0.42 ± 0.12 mmol/L). Technetium-99m octreotide scintigraphy was performed in 11 patients and showed lesions in the right distal femur, left femoral head, and right tibial plateau, respectively, in three patients. Magnetic resonance imaging (MRI) was negative for lesions in one patient. Two patients underwent biopsies that showed negative histopathology. Two patients, at 2 years and 8 months, respectively, after having negative technetium-99m octreotide studies, underwent 18F-fluorodeoxyglucose positron emission tomography/computed tomography (CT), which revealed lesions in the sacrum and soft tissue of the left palm, respectively. One tumor was detected by CT and MRI. Overall, lesion sites were the head (two patients, 16.7%), thoracic and lumbar region (two, 16.7%), pelvis (three, 25%), lower limbs (four, 33.3%), and upper limbs (one, 8.3%). All patients underwent surgery, and histopathology showed phosphaturic mesenchymal tumors in each. Postoperatively, serum phosphorus concentrations normalized within 2-7 days in 11 patients. With follow-ups of 1-41 months, surgery was effective in 10 patients. One patient developed local recurrence and another had metastases. CONCLUSIONS: Locating tumors responsible for tumor-induced osteomalacia is often challenging. Although complete tumor resection confers a good prognosis in most patients, surveillance for recurrence and metastasis is necessary. Before surgery or when surgery is not indicated, oral phosphate can alleviate symptoms and metabolic imbalance.

[Aggressiveness and extent of prostatic inflammation relates with serum PSA levels in type IV prostatitis].

OBJECTIVE: To investigate the relationship of the histopathologic grade and extent of prostatic inflammation with the level of serum PSA in patients with type IV prostatitis. METHODS: We performed transrectal ultrasound-guided prostate biopsy for 120 patients suspected of prostate cancer and included in this study only those with benign prostate hyperplasia (BPH) and prostatitis (n = 46), excluding the cases with prostate cancer and those with BPH but no prostatitis. We evaluated the relationship between prostatic inflammation and serum PSA levels based on the three-grade pathohistologic criteria for the extent, location and aggressiveness of prostatic inflammation. The serum tPSA levels, fPSA levels, % fPSA, and PSAD were compared among different groups. RESULTS: As for the extent of inflammation, 35 of the 46 included cases were grade I (tPSA: [8.46 +/- 4.09] microg/L; fPSA: [1.75 +/- 0.93] microg/L; PSAD: 0.15 +/- 0.11), 7 were grade II (tPSA: [15.26 +/- 5.26] microg/L; fPSA: [2.54 +/- 0.72] microg/L; PSAD: 0.26 +/- 0.07) and 4 were grade III (tPSA: [21.05 +/- 7.58] microg/L; fPSA: [3. 19 +/- 1.13] microg/L; PSAD: 0.42 +/- 0.19), with statistically significant differences among the three groups in the levels of tPSA (P = 0.001), fPSA (P = 0.008) and PSAD (P < 0.001). Regarding the location of inflammation, 19 cases were grade I, 17 were grade II and 10 were grade II, with no significant differences in tPSA, fPSA and %fPSA among the three grades (P > 0.05). As for the aggressiveness of inflammation, 32 cases were grade I (tPSA: [8.37 +/- 4.07] microg/L; fPSA: [1.76 +/- 0.93] microg/L; PSAD: 0.14 +/- 0.11), 10 were grade II (tPSA: [13.30 +/- 5.69] microg/L; fPSA: [3.27 +/- 2.21] microg/L ; PSAD: 0.25 +/- 0.06) and 4 were grade III (tPSA: [21.05 +/- 7.58] microg/L; fPSA: [3.19 +/- 1.13] microg/L; PSAD: 0.42 +/- 0.19), with statistically significant differences among the three grades in the levels of tPSA (P = 0.002), fPSA (P = 0.024) and PSAD (P < 0.001). The extent of inflammation was positively correlated with the levels of tPSA (r = 0.6, P < 0.001), fPSA (r = 0.5, P = 0.001) and PSAD (r = 0.6, P < 0.001), and so was the aggressiveness of inflammation (tPSA: r = 0.5, P < 0.001; fPSA: r = 0.4, P = 0.008; PSAD: r = 0.7, P < 0.001), but a negative correlation was found between the aggressiveness of inflammation and %fPSA (r = -0.4, P = 0.013). CONCLUSION: The aggressiveness and extent of prostatic inflammation in asymptomatic prostatitis patients are significantly correlated with the level of serum PSA, which may help pathologists to avoid unnecessary repeated biopsies for patients with high-grade prostatitis.

A novel FGF23 mutation in hyperphosphatemic familial tumoral calcinosis and its deleterious effect on protein O-glycosylation.

Background: Hyperphosphatemic familial tumoral calcinosis (HFTC) is a rare disease characterized by hyperphosphatemia and ectopic calcification, predominantly at periarticular locations. This study was performed to characterize the clinical profile of tumoral calcinosis and to identify gene mutations associated with HFTC and elucidated its pathogenic role. Methods: The three subjects (two male and one female) were aged 30, 25 and 15 years, respectively. The clinical features, histopathological findings, and outcomes of three subjects with HFTC were retrospectively reviewed. The three subjects were analyzed for FGF23, GALNT3 and KL mutations. Function of mutant gene was analyzed by western blotting and wheat germ agglutinin affinity chromatography. Results: All subjects had hyperphosphatemia and elevated calcium-phosphorus product. Calcinosis positions included the left shoulder, left index finger, and right hip. Bone and joint damage were present in two cases and multiple foci influenced body growth in one case. The histopathological features were firm, rubbery masses comprising multiple nodules of calcified material bordered by the proliferation of mononuclear or multinuclear macrophages, osteoclastic-like giant cells, fibroblasts, and chronic inflammatory cells. The novel mutation c.484A>G (p.N162D) in exon 3 of FGF23 was identified in one subject and his family members. Measurement of circulating FGF23 in the subject confirmed low intact FGF23 and increased C-terminal fragment. In vitro experiments showed that the mutant FGF23 proteins had defective O-glycosylation and impaired protein proteolysis protection. Conclusion: We identified a novel FGF23 missense mutation, and confirmed its damaging role in FGF23 protein O-glycosylation. Our findings expand the current spectrum of FGF23 variations that influence phosphorus metabolism.

[Clinicopathologic features of primary osteosarcoma in elderly patients].

OBJECTIVE: To study the clinical manifestations, radiologic findings, pathologic diagnosis and differential diagnosis of primary osteosarcoma in elderly patients. METHODS: Twelve cases of primary osteosarcoma occurring in patients older than 60 years were encountered during the period from 1985 to 2010. The clinical manifestations, radiologic features and pathologic findings were studied and the follow-up data were analyzed. RESULTS: The sites of involvement included long bones (number = 7), ilium (number = 1), craniofacial bones (number = 2) and soft tissue (number = 2). Radiologic examination showed a mixture of osteosclerotic and osteolytic lesions in 10 patients, soft tissue lesions with high-density areas in 2 patients and soft tissue lesions with periosteal reaction in 8 patients. Histologically, most cases showed features of conventional osteosarcoma. There were 2 cases of malignant fibrous histiocytoma-like osteosarcoma, 2 cases of chondroblastic osteosarcoma and 1 case of well-differentiated intraosseous osteosarcoma. Immunohistochemical study played little role in pathologic diagnosis. Ten patients had undergone amputation, including one patient who had received adjuvant chemotherapy beforehand. Nine patients had follow-up information available. Three of them died of lung metastasis and 1 died of cardiovascular disease. CONCLUSIONS: Primary osteosarcoma rarely occurs in elderly patients and can easily be missed. Correlation with clinical, radiologic and histologic features is important for arriving at a correct diagnosis.

Histopathology Findings of the Lunate in Stage III Kienböck's Disease.

Background: The etiology and natural history of Kienböck's disease remain unclear. Traditionally it has been defined as avascular necrosis of the lunate. The authors tried to demonstrate different tissue distribution, the area ratio of necrotic tissue and vessel counts inside the whole Kienböck lunate to reveal a dynamic process of the lunate collapse. Methods: Five lunates from patients with stage III Kienböck's disease and one cadaveric lunate not involved by Kienböck's disease were sampled. They were sectioned, H&E stained, and evaluated. The thickness of trabecular bone and the area of necrotic tissue were measured with Image-Pro Plus. The number of vessels was counted manually. Results: In the normal lunate, the bone trabeculae showed a uniform distribution with fatty marrow filled the interspace between the trabeculae. In the lunates with Kienböck disease, the trabeculae fracture and necrosis located in the central part with massive fibrous granular tissue proliferation. There were also some chondroid metaplasia at the palmar and dorsal ends. The trabeculae of the lunates of the Kienböck's disease [0.188 mm (0.153 mm, 0.236 mm)] was significantly thicker than the normal lunates [0.146 mm (0.124 mm, 0.164 mm)]. The necrosis was localized around the fracture sites instead of the whole lunate. The mean necrosis area only accounts for 16.3% ± 8.9% of the whole section. Such kind of focal necrosis is quite similar to those around the traumatic fracture ends of other bones. Even in stage III Kienböck lunates, the vessels are quite abundant (221 ± 42 in one sagittal section), while the vessels inside the normal lunate were 352 ± 28. Conclusions: There is neither massive nor obvious generalized avascular bone necrosis in our histopathology observations. The focal necrosis and vessel damage were more likely associated with the broken trabeculae inside the lunate. Based on our histopathology observations, we suggested that the progressive process of Kienböck's disease could be described as lunate nonunion advanced collapse instead of avascular necrosis.

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice.

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

[Application of the in situ hybridization with EWS dual-color break-apart fluorescence probe and anti-CD99 and anti-FLI-1 antibodies in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor].

OBJECTIVE: To investigate the value of using EWS dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique, CD99 and FLI-1 antibodies immunohistochemistry in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumor (EWS/PNET). METHODS: Thirty-five cases of EWS/PNET and 24 cases of non-EWS/PNET small round cell tumor were analyzed by FISH and immunohistochemically detected with FLI-1 and CD99 antibodies. Comparison between FISH and immunohistochemical results was carried out and their diagnostic value was evaluated. RESULTS: The sensitivity, specificity, positive predictive value and negative predictive value of FISH in EWS/PNET were 93.8%(30/32), 81.8%(18/22), 88.2%(30/34) and 90%(18/20); those of CD99 were 100%(35/35), 58.3% (14/24), 77.8%(35/45) and 100%(14/14); those of FLI-1 were 71.4%(25/35), 62.5%(15/24), 73.5%(25/34), 60%(15/25), respectively. The sensitivity, specificity, positive predictive value and negative predictive value of combined use of CD99 and FLI-1 were 71.4%(25/35), 75%(18/24), 80.6%(25/31) and 64.3%(18/28), those of combined use of FLI-1 and FISH were 68.7%(22/32), 86.4%(19/22), 88%(22/25) and 65.5%(19/29), those of combined use of CD99 and FISH were 93.7%(30/32), 95.5%(21/22), 96.8%(30/31) and 91.3%(21/23), respectively. CONCLUSION: EWS dual-color, break-apart rearrangement probe FISH is a highly sensitive and specific technique in the diagnosis of EWS/PNET. The combination of CD99 and FISH is the method of choice for the diagnosis of EWS/PNET. The combination of CD99 and FLI-1 can improve the specificity in EWS/PNET without the data of FISH.

Transition from Tumor Tissue to Bone Marrow in Patients with Appendicular Osteosarcoma after Neoadjuvant Chemotherapy.

BACKGROUND:: Limb-salvage surgery is the standard procedure for the treatment of appendicular osteosarcoma. Precise resection is the trend in limb-salvage surgery. The aim of this study was to evaluate a large series of cases to identify the histological relationship between the tumor and marrow and determine the intramedullary transition type and width from the tumor to normal marrow in patients with osteosarcoma after neoadjuvant chemotherapy. METHODS:: One hundred and six osteosarcoma specimens were evaluated. The tissue specimens were sectioned through the coronal axis by an electronic saw. The tissue was immersed in formalin solution for fixation and subsequently decalcified. The interface between the tumor and normal bone marrow was grossly determined and submitted for microscopic evaluation to detect the relationship between the tumor and bone marrow and identify the transition type and width. All histological slides were examined by experienced orthopedic pathologists. RESULTS:: Histologically, the interface between the tumor and normal bone marrow was classified into two patterns: "clear" and "infiltrated." The clear pattern, characterized by a clear boundary between the tumor and marrow, was identified in sixty cases (56.6%). A subtype of the clear type, characterized by fibrous bands between the tumor and marrow, was found in 13 cases (12.3%). The infiltrated pattern, characterized by a boundary with tumor cell clusters embedded in the marrow, was found in 46 cases (43.4%). The infiltrating depth varied from 1 to 4 mm (mean, 2.6 ± 0.7 mm). No tumor cells were observed in the normal bone marrow areas next to the interface. CONCLUSIONS:: The transition from osteosarcoma tissue to bone marrow after neoadjuvant chemotherapy can be divided into two histological patterns: clear and infiltrated. The greatest infiltration width was 4 mm from tumor to normal marrow in this study. This depth should be considered in the presurgical plan.

Anti-osteoporosis effect of Epimedium via an estrogen-like mechanism based on a system-level approach.

ETHNOPHARMACOLOGICAL RELEVANCE: Epimedium is a popular traditional herbal medicine worldwide that has long been used to relieve osteoporosis. The estrogenic properties of the herb are conferred by several phytoestrogens, such as flavonoids, lignans, and steroids. However, the poor understanding on the estrogen-like mechanism of Epimedium at the molecular and system levels limits the applications of this herb in osteoporosis treatment. MATERIALS AND METHODS: In this study, systems pharmacology was established to investigate the relationship between Epimedium and estrogen against osteoporosis by integrating active component screening, drug-likeness evaluation, herb feature mapping, target prediction and validation, and network analysis. RESULTS: A total of 77 active components that possessed similar structural features to estrogen as determined using herb feature mapping were selected from Epimedium by oral bioavailability prediction and drug-likeness evaluation. Twenty three osteoporosis-related targets were obtained from the active components of Epimedium as potential targets, 11 of which were common targets with estrogen. All osteoporosis-related targets were further mapped to compound-target and target-pathway networks. Results displayed that Epimedium can exert anti-osteoporosis effects by directly regulating the 11 estrogen-related targets and a set of target proteins on five estrogen-related pathways. CONCLUSION: This study explained the estrogen-like mechanism of Epimedium in preventing and treating osteoporosis, and provided a new standpoint for exploring the traditional herbal medicine against osteoporosis.

Pleural Effusion in Multiple Myeloma.

OBJECTIVE: Pleural effusion is rarely observed in patients with multiple myeloma (MM). Myeloma cell infiltration or invasion to the pleura is very rare. This study aimed to investigate the clinical characteristics of pleural effusion in patients with MM. METHODS: We retrospectively reviewed the medical records of patients diagnosed with pleural effusion, MM, and pleural effusion with MM between 2004 and 2014 at Beijing Jishuitan Hospital. The present study included patients with pleural effusion who underwent cytological, bacteriological, biochemical and other testing. The cytopathology of abnormal pleural effusion cells was not diagnostic, thus flow cytometry was performed. MM was defined using the diagnosis standard of NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) 2014 for MM. RESULTS: This study included 3,480 pleural effusion patients and 319 MM patients. There were 34 patients with both MM and pleural effusion (17 men and 17 women). The average age was 63 years (range, 48-84 years). Pleural effusion with MM was caused by congestive heart disease, chronic renal failure, hypoalbuminemia, pulmonary infarctions, cirrhosis, pulmonary arterial hypertension, parapneumonic effusion, tuberculous pleural effusion, and myelomatous pleural effusion (MPE). The diagnosis of MPE was confirmed by the detection of myeloma cells in the pleural fluid using flow cytometric analyses. There were only 2 MPE cases in our study. The first MPE case was a woman. The first clinical manifestation was pleural effusion, and the diagnosis was non-secretory MM, DSS stage IIIA (Durie-Salmon staging system); ISS stage I (the International Staging System). The second MPE case was a man who was diagnosed with MM IgA-κ, DSS stage IIIA; ISS stage II. CONCLUSION: The detection rate of MPE was very low. MPE tended to present with yellow exudates and the lack of physical and chemical characteristics. Furthermore, patients with MPE exhibited many yellow nodules on the pleura. These nodules were lobulated and had abundant blood supply. The routine pleural effusion pathological examination had low sensitivity. Flow cytometry may be more useful for improving the detection rate of MPE.

[Development of an automatic vacuum liquid chromatographic device and its application in the separation of the components from Schisandra chinensis (Turz) Baill].

In order to meet the needs of efficient purification of products from natural resources, this paper developed an automatic vacuum liquid chromatographic device (AUTO-VLC) and applied it to the component separation of petroleum ether extracts of Schisandra chinensis (Turcz) Baill. The device was comprised of a solvent system, a 10-position distribution valve, a 3-position changes valve, dynamic axis compress chromatographic columns with three diameters, and a 10-position fraction valve. The programmable logic controller (PLC) S7- 200 was adopted to realize the automatic control and monitoring of the mobile phase changing, column selection, separation time setting and fraction collection. The separation results showed that six fractions (S1-S6) of different chemical components from 100 g Schisandra chinensis (Turcz) Baill. petroleum ether phase were obtained by the AUTO-VLC with 150 mm diameter dynamic axis compress chromatographic column. A new method used for the VLC separation parameters screened by using multiple development TLC was developed and confirmed. The initial mobile phase of AUTO-VLC was selected by taking Rf of all the target compounds ranging from 0 to 0.45 for fist development on the TLC; gradient elution ratio was selected according to k value (the slope of the linear function of Rf value and development times on the TLC) and the resolution of target compounds; elution times (n) were calculated by the formula n ≈ ΔRf/k. A total of four compounds with the purity more than 85% and 13 other components were separated from S5 under the selected conditions for only 17 h. Therefore, the development of the automatic VLC and its method are significant to the automatic and systematic separation of traditional Chinese medicines.

Systems pharmacology-based drug discovery for marine resources: an example using sea cucumber (Holothurians).

ETHNOPHARMACOLOGICAL RELEVANCE: Sea cucumber, a kind of marine animal, have long been utilized as tonic and traditional remedies in the Middle East and Asia because of its effectiveness against hypertension, asthma, rheumatism, cuts and burns, impotence, and constipation. In this study, an overall study performed on sea cucumber was used as an example to show drug discovery from marine resource by using systems pharmacology model. The value of marine natural resources has been extensively considered because these resources can be potentially used to treat and prevent human diseases. However, the discovery of drugs from oceans is difficult, because of complex environments in terms of composition and active mechanisms. Thus, a comprehensive systems approach which could discover active constituents and their targets from marine resource, understand the biological basis for their pharmacological properties is necessary. MATERIALS AND METHODS: In this study, a feasible pharmacological model based on systems pharmacology was established to investigate marine medicine by incorporating active compound screening, target identification, and network and pathway analysis. RESULTS: As a result, 106 candidate components of sea cucumber and 26 potential targets were identified. Furthermore, the functions of sea cucumber in health improvement and disease treatment were elucidated in a holistic way based on the established compound-target and target-disease networks, and incorporated pathways. CONCLUSIONS: This study established a novel strategy that could be used to explore specific active mechanisms and discover new drugs from marine sources.