Two classic OBPs modulate the responses of female Holotrichia oblita to three major ester host plant volatiles.

Insects possess a fairly sophisticated olfactory system in their antennae to detect odorants essential for their survival and reproduction. Among them, insect first perceives odour sources by odorant-binding proteins (OBPs) to locate host-plants. Methyl salicylate, (Z)-3-hexenyl acetate and dibutyl phthalate are major volatile components of Ulmus pumila and Ricinus communis and elicit strong responses of the scarab beetle Holotrichia oblita adults. However, olfactory perception of the scarab beetle to these odorant compounds is unclear. In the current study, we cloned the OBP6 and OBP7 of H. oblita. The expression pattern shows that the two genes were highly expressed in the antennae of female beetles. Binding assays verified that the HoblOBP6 had a better binding affinity to methyl salicylate, and so did HoblOBP7 to (Z)-3-hexenyl acetate and dibutyl phthalate. The effect on the responses of female beetles to the three compounds was decreased significantly after these two genes were silenced by RNA interference. These results indicate that HoblOBP6 and HoblOBP7 are essential for female H. oblita perception of methyl salicylate, (Z)-3-hexenyl acetate and dibutyl phthalate. Our study provides important insights into the olfactory mechanism of female H. oblita to ester plant volatiles and could facilitate the development of potential pest control strategies in the field.

[Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm].

Objective: To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient, and to study its biological characteristics, genomic information, and effects on bacterial biofilm. Methods: (1) In 2018, pan-drug resistant Klebsiella pneumoniae UA168 (hereinafter referred to as the host bacteria) solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Ruijin Hospital) was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method, drip plate method, and double-agar plate method. The bacteriophage was named as phage KP168 and the plaque morphology was observed. (2) The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis, and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining. (3) The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients' blood in Ruijin Hospital by the drip plate method, and then the lysis rate was calculated. (4) The phage KP168 solution at a initial titer of 9.3×10(11) plaque-forming unit (PFU)/mL (400 µL per tube) and the host bacteria solution at a concentration of 1×10(9) colony-forming unit (CFU)/mL (4 mL per tube) were conventionally shaking cultured together for 4 hours at multiplicity of infection (MOI) of 10.000, 1.000, 0.100, 0.010, or 0.001, respectively (1 tube per MOI). The titer of phage KP168 was measured by the double-agar plate method (the measurement method was the same below) to select the optimal MOI. The experiment was repeated three times. (5) The host bacteria solution at a concentration of 1×10(9) CFU/mL (4 mL per tube) and the phage KP168 solution at an adjusted titer of 5×10(7) PFU/mL (400 µL per tube) were mixed at the MOI of 0.005. The plaques were counted 0 (immediately), 1, 2, 3, 4, 5, 15, and 30 minutes (1 tube at each time point) after mixing by the double-agar plate method (the counting method was the same below), and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time. The experiment was repeated three times. (6) The host bacteria solution at a concentration of 1×10(9) CFU/mL (300 µL per tube) and the phage KP168 solution at a titer of 5×10(8) PFU/mL (60 µL per tube) were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time. The phage KP168 titer was measured 0 (immediately), 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 minutes after culture, and a one-step growth curve was drawn. The experiment was repeated three times. (7) The phage KP168 solution at a titer of 2.5×10(10) PFU/mL was left to stand for 1 hour at 37, 40, 50, 60, or 70 ℃ (3 tubes at each time point, 1 mL per tube) for counting the plaques, and then the thermal stability curve was drawn. SM buffer at a pH values of 5.0, 6.0, 7.0, 7.4, 8.0, 9.0, or 10.0 were added to the phage KP168 solution at a titer of 3.0×10(10) PFU/mL, respectively. The mixed solution was left to stand for 1 hour at 37 ℃ (3 tubes of each pH, each tube containing 100 µL phage KP168 solution and 900 µL SM buffer), and then the plaques were counted, and an acid-base stability curve was drawn. (8) The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment (2). The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168. Nucleotide's BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence, and Blastx function was used to translate the coding sequence into protein for its function prediction. The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded. (9) In a 96-well plate, at a MOI of 1.000, 0.100, 0.010 or 0.001 (3 wells per MOI), 20 µL phage KP168 solution at a initial titer of 5.8×10(10) PFU/mL was added to 200 µL host bacteria solution at a concentration of 1.5×10(8) CFU/mL (the same concentration below) for co-cultivation for 48 hours. After 200 µL host bacteria solution was left to stand for 48 hours, 20 µL phage KP168 solution at a titer of 1×10(6,) 1×10(7,) 1×10(8,) 1×10(9,) or 1×10(10) PFU/mL (3 wells per titer) was added respectively for action for 4 hours. In both experiments, 200 µL host bacteria solution added with 20 µL SM buffer (3 wells) acted as a negative control, and 220 µL LB culture medium (3 wells) acted as a blank control. Absorbance values were measured by a microplate reader, and inhibition/destruction rates of biofilm were calculated. The experiments were both repeated three times. Results: (1) The plaques of phage KP168 successfully isolated and purified were transparent and round, and its diameter was approximately 1.5 mm. (2) The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail. (3) The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients, with a lysis rate of 65.0%. (4) When MOI was 1.000, the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours, which was the optimal MOI. (5) After the mixing of the phage KP168 with the host bacteria for 4 minutes, the percentage of the adsorbed phage reached the highest, which was the optimal adsorption time. (6) The one-step growth curve showed that during the lysis of the host bacteria by phage KP168, the incubation period was about 10 minutes, and the lysis period was about 40 minutes. (7) With the condition of 40 ℃ or pH 7.4, the number of plaques and the activity of phage KP168 reached the highest. (8) The genome of phage KP168 was a linear double-stranded DNA with a length of 40 114 bp. There were 48 possible coding sequences. It had the highest similarity to Klebsiella phage_vB_Kp1. The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions. No resistance genes or virulence factor genes were found. The GeneBank accession number was KT367885. (9) After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI, the inhibition rates of biofilm were similar, with an average of about 45%. After the phage KP168 with a titer of 1×10(9) PFU/mL acted on the biofilm formed by the host bacteria for 4 h, the destruction rate of biofilm was the highest, reaching an average of 42%. Conclusions: In this study, a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient, phage KP168, is isolated from sewage, which belongs to the tailless phage. It has a wide host spectrum, short adsorption time, and short incubation period, with certain thermal and acid-base stability. Its genomic information is clear, and it does not contain resistance genes or virulence factor genes. It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.

The study of microanatomy of intestinal epithelium in the Chinese soft-shelled turtle (Pelodiscus sinensis).

The microanatomy of the intestinal epithelium in the Chinese soft-shelled turtle (CST) was studied by light and transmission electron microscopy (TEM). The small intestinal epithelium (SIE) was single layered or pseudostratified. The enterocytes contained mitochondria or mitochondria and lipid droplets. The enterocytes were arranged tightly in the apical parts of epithelium and connected by desmosomes and interdigitations. The large intestinal epithelium (LIE) was pseudostratified and the enterocytes did not contain lipid droplets. Enterocytes were arranged compactly in the apical part, forming spaces in the middle and basal parts of epithelium. Numerous mucous cells were scattered in the epithelium and there were intraepithelial lymphocytes (IELs) with their pseudopodia extended into the intestinal lumen. This study provides detailed features of intestinal epithelium in the Pelodiscus sinensis that could be related to function. In addition, these findings are discussed in relation to other vertebrates.

Zhi-Long-Huo-Xue-Tong-Yu modulates mitochondrial fission through the ROCK1 pathway in mitochondrial dysfunction caused by streptozotocin-induced diabetic kidney injury.

Zhi-Long-Huo-Xue-Tong-Yu (ZLHXTY) is a defined mixture of 5 herbs developed by Professor S.J. Yang according to the Buyang Huanwu decoction method, which has been recorded in the Yilingaicuo. This study investigated the renoprotective effects of ZLHXTY on mitochondrial dysfunction induced by diabetic kidney injury in a diabetic rat model. Diabetes was induced by a single intravenous injection of streptozotocin. Rats were daily fed either ZLHXTY or vehicle beginning in the 1st week after injection. Levels of mitofusin 2 (mfn2), dynamin-related protein 1 (Drp1), caspase-9, and rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) were detected using Western blotting. Levels of intracellular calcium and adenosine triphosphate (ATP) were examined using an enzyme-linked immunosorbent assay. An electron microscopic examination of kidney tissue was performed. The levels of mfn2 and ATP in the diabetes and ZLHXTY groups decreased from the 4th week after modeling. The expression levels of Drp1, ROCK1, and caspase-9 increased in the diabetes group but decreased in the ZLHXTY group from the 4th week after modeling. Compared with the diabetes group, ZLHXTY treatment decreased the mesangial expansion index and proteinuria levels, and improved the pathological changes typical of diabetic kidney injury. Furthermore, ZLHXTY treatment inhibited the activation of ROCK1 and expression of Drp1 and caspase-9, but did not affect the expression of mfn2. This study indicates that ZLHXTY treatment could protect kidney tissue from diabetic injury through the ROCK1 pathway response to mitochondrial dysfunction induced by diabetes.

[Tongue tip microcirculation in patients with different symptoms].

Observations of tongue tip microcirculation were made on 104 patients with different symptom-complex [5 groups: Yin deficiency, Yang deficiency, Qi.blood deficiency, Qi stagnation-blood stasis, damp-heat] in view of TCM, and on 100 healthy persons. 10 indicators including the transverse diameter of the fungiform papillae, morphology of microvascular clumps in the tongue papillae, congestion of the top of microvascular loop, loop dilation, blood color, hemodynamics in microvascular loop, exudation, hemorrhage and loop morphology were observed. The results were: patients of each group were found to have to different degree microcirculation dysfunction; every group differed with Qi stagnation-blood stasis group of being most different. The numbers of abnormal indicators: Qi stagnation-blood stasis group had all 10; Yang deficiency group 9; Yin deficiency and Qi-blood deficiency group 8, 8 respectively; damp-heat group 7. This study discussed the relationship between tongue tip microcirculation of each group and the changes of tongue picture and typing of differentiation of symptoms and signs in view of TCM.

[Alkaloids with cardiovascular activities--synthesis of protoberberine].

It has been reported that berberine (BR) has positive inotropic and negative chronotropic effects. Recently, it has been tried in patients with arrhythmia and congestive heart failure. But BR, a quaternary ammonium salt, is absorbed poorly. We took BR as leading compound to synthesize its derivatives in order to find orally active agents. Compounds substituted by -OCH3 in different positions of the D ring were synthesized. In the screening of inhibition of electrically induced contraction of isolated guinea pig left atria and spontaneous beating of its right atria, the synthesized compounds were found to possess activity of inhibiting spontaneous beating of isolated guinea pig right atria. Compound I4 was shown to be the most effective of the synthesized compounds with IC50 value of 4.34 (mumol). The IC50 value of BR was 204 (mumol).

[Differential diagnosis in the preliminary evaluation of esterase responses of tongue coating cells].

The tongue coating cells were stained by Alpha-naphthol acetate esterase and naphthol AS-D chloroacetate esterase in 100 patients with different symptom-complex [five groups: dampheat, damp-heat due to blood stasis, Yin deficiency, Yang deficiency and Qi-blood deficiency] in view of TCM, and in 116 healthy persons. The responses of the epithelial cells of the tongue and the leukocytes to the two esterase stainings were observed. The results were: the number of nonspecific esterase was much higher in the patients, especially in the patients with Yin or Qi-blood deficiency, than that in the healthy persons (P less than 0.001); responses to ASDCE suggested that the leukocytes of the patients were different from those of the healthy persons in classification, more granulocytes in the healthy persons, and increased proportion of monocytes and lymphocytes in the patients, the most increased proportion of monocytes in the patients with Qi-blood deficiency and the most increased proportion of lymphocytes in the patients with Yang deficiency. The mechanisms of the different responses to the two esterase stainings in the patients and healthy persons, and the relationship between these mechanisms and the symptom-complexes by TCM were discussed.