Integrating tumor mutational burden and transcriptome expression into prediction of immune checkpoint inhibitor response and prognosis of patients with colon cancer.

Whether tumor mutational burden (TMB), which refers to the total number of somatic or acquired mutations per million bases in a particular region of the tumor genome, can serve as a predictive biomarker of immune checkpoint inhibitor (ICI) therapy for colon cancer remains unclear. Hereby, we retrospectively investigated the differentially expressed genes (DEGs) based on the level of TMB and tried to established a risk score model as a novel biomarker. The DNA mutation data were retrieved from the Masked Somatic Mutation in Genomic Data Commons data portal of the Cancer Genome Atlas, where the RNA sequencing data, clinical information, and survival outcomes of patients were downloaded. Patients with incomplete clinical information were excluded. The immune score and stromal score were calculated to investigate immune infiltration. The patients were grouped into TMB-high group and the TMB-low group based on the median value of TMB. An immune relevant gene set was obtained from the Immunology Database and Analysis Portal to identify immune-related DEGs. The Cox proportional hazard model and nomogram were applied to establish the risk model. In results: the TMB value was associated with age (p≤0.001), clinical stage (p≤0.001), N stage (p≤0.001), M stage (p=0.003), and immune score (p≤0.001). Twenty-nine immune-related DEGs were identified as enriched in immune response-related function or pathway and tumorigenesis signaling. Nine of 29 were determined to establish a riskScore model. The riskScore suggested a positive relationship with the TMB value (p=0.033), immune score (p≤0.001), and tumor immune dysfunction and exclusion (TIDE) (p=0.002) and presented an independent prognostic factor (p≤0.001, HR=1.04), which predicted the overall survival with good specificity. We concluded that the combination of TMB with transcriptome expression has a predictive and prognostic value for patients treated with ICIs.

Comparison between a novel human cortical bone screw and bioabsorbable interference screw for graft fixation of ACL reconstruction.

OBJECTIVE: To compare the mechanical behavior of a novel bioabsorbable cortical interference screw (BCIS) with bioabsorbable interference screw (BIS; Polylactate hydroxyapatite) used for anterior cruciate ligament (ACL) reconstruction in femoral and tibial fixation with doubled Achilles tendon graft in vitro. PATIENTS AND METHODS: 30 paired goat knee specimens were harvested from 15 male sheep aged 18 months. All soft tissues were stripped from the bones of 20 paired specimens, and the last 10 paired specimens were stripped all soft tissues besides ACL (femur-ACL-tibia complex). The Achilles tendon was harvested as graft for ACL reconstruction. The specimens were divided into several groups: BCIS femoral fixation (group A, n=10), BIS femoral fixation (group B, n=10), BCIS tibial fixation (group C, n=10), BIS tibial fixation (group D, n=10), Group E is femur-ACL-tibia complex (n=10). Cyclic loading test was performed from 50 to 250 N at 1 Hz for 1000 cycles and followed by a load-to-failure test at 25 mm/sec. A paired t-test was used to compare the biomechanical properties of group A, B, E and group C, D, E. RESULTS: No fixation structures failed during the cyclic phase. Cyclic displacement for group B was superior to group A, and showed statistically significant difference after 30, 100, 500, 1000 cycles. Group E got minimum cyclic displacements compared with group A and group B, and showed statistically significant difference after 500, 1000 cycles compared with group A. Cyclic displacement for group D was superior to group C, and showed statistically significant difference after 100, 500, 1000 cycles. Group E got minimum cyclic displacements compared with group C and group D, and showed statistically significant difference after 500,1000 cycles compared with group C. Regarding MFL, group A was superior to group B (572.10±111.12 N vs. 413.96±34.56 N, p=0.118), group E was superior to group A (599.74±85.45N vs. 572.10±111.12 N, p=0.992), and group C was superior to group D (802.88±240.07 N vs. 415.63±51.9 N, p<0.001), group C was superior to group E (802.88±240.07 N vs. 599.74±85.45 N, p=0.024). Regarding YL, group A was superior to group B (521.57±93.96 N vs. 366.99±44.66 N, p=0.109), group E was superior to group A (565.37±66.05 N vs. 521.57±93.96 N, p=0.952), and group C was superior to group D (735.63±242.91 N vs. 394.49±31.90 N, p<0.001), group C was superior to group E (735.63±242.91 N vs. 565.37±66.05 N, p=0.063). Regarding stiffness, group A was superior to group B (157.36±34.31 N/mm vs. 91.98±25.57 N/mm, p=0.001), group E was superior to group A (181.35±25.42 N vs. 157.36±34.31 N/mm, p=0.529), and group C was superior to group D (175.28±43.19 N/mm vs. 128.24±18.92 N/mm, p=0.032), group E was superior to group C (181.35±25.42 N/mm vs. 175.28±43.19 N/mm, p=0.995). CONCLUSIONS: In vitro, this experimental study suggested the biomechanical properties of novel bioabsorbable cortical interference screw (BCIS) were superior to bioabsorbable interference screw (BIS) used for femoral and tibial anterior cruciate ligament (ACL) reconstruction in a goat knee model.

Heterogeneous to homogeneous melting transition visualized with ultrafast electron diffraction.

The ultrafast laser excitation of matters leads to nonequilibrium states with complex solid-liquid phase-transition dynamics. We used electron diffraction at mega-electron volt energies to visualize the ultrafast melting of gold on the atomic scale length. For energy densities approaching the irreversible melting regime, we first observed heterogeneous melting on time scales of 100 to 1000 picoseconds, transitioning to homogeneous melting that occurs catastrophically within 10 to 20 picoseconds at higher energy densities. We showed evidence for the heterogeneous coexistence of solid and liquid. We determined the ion and electron temperature evolution and found superheated conditions. Our results constrain the electron-ion coupling rate, determine the Debye temperature, and reveal the melting sensitivity to nucleation seeds.

Epigenetic mechanisms contribute to decrease stearoyl-CoA desaturase 1 expression in the liver of dairy cows after prolonged feeding of high-concentrate diet.

Subacute ruminal acidosis (SARA) of dairy cattle is a widely occurring but not very overt metabolic disorder thought to impair milk composition. The enzyme stearoyl-CoA desaturase 1 (SCD1) is rate-limiting for the formation of Δ-9 unsaturated fatty acids and thus crucially involved in controlling lipid metabolism in the liver. It is known that SCD1 expression is downregulated during SARA, but the underlying molecular mechanisms are unknown. To study these mechanisms, we enrolled 12 healthy multiparous mid-lactation Holstein cows into a diet-induced SARA experiment. Six cows were fed a high-concentrate diet for 18 weeks (60% content of high-concentrate to 40% forage; HC group), whereas the others received a low-concentrate diet ad libitum (40% high-concentrate content to 60% forage; LC group). Sustained low ruminal pH values (pH 5.6 maintained for 4 h/d) and reduced milk yield performance (2.07 kg/d less than LC cows) verified that SARA had been induced in the HC group. Results showed a significantly decreased concentrations of cis-9 monounsaturated long-chain fatty acids in plasma collected from hepatic but not portal veins. This was matched by reduced SCD1 mRNA and protein concentrations in HC livers. The expression levels of genes related to lipid formation (DGAT1 and PLIN2) were downregulated during SARA, whereas those of catabolic genes (CPT1A, CPT2, and ACOX1) and some inflammatory genes were upregulated. Expression of SCD1 was downregulated through reduced transcription and abundance of the transcription factor sterol regulatory element-binding protein 1 (SREBP1c).This effect was augmented by local chromatin tightening and DNA methylation at and around the SREBP1c binding site in the SCD1 promoter. Chromatin immunoprecipitation assays confirmed that SARA reduced SREBP1c binding at the SCD1 promoter; hence, epigenetic mechanisms are involved in regulating the expression of genes related to long-chain fatty acid modification, partially through downregulation of both SCD1 and SREBP1c in the liver. Our results suggest that in addition to inflammatory genes, SCD1 is also involved in SARA-induced epigenetic regulation and its associated metabolic changes. This knowledge might help to provide a target for intervening against the detrimental metabolic effects of SARA.

Lipopolysaccharide derived from the digestive tract provokes oxidative stress in the liver of dairy cows fed a high-grain diet.

The aims of this study were to measure oxidative stress parameters and to investigate the molecular mechanism triggered by grain-induced subacute ruminal acidosis in mid-lactation cows. Twelve Holstein-Friesian cows with an average weight of 455±28kg were divided into 2 groups and subjected to 2 diets over 18wk: either a low-grain (forage-to-concentrate ratio=6:4) or a high-grain (forage-to-concentrate ratio=4:6) diet based on dry matter. Being fed a long-term high-grain diet resulted in a significant decrease in rumen pH and a significant increase in ruminal lipopolysaccharide (LPS) at 4 h postfeeding in the morning. The increase was also observed in LPS concentrations in the portal vein, hepatic vein, and jugular vein blood plasma as well as reduced milk yield in a high-grain diet. Cows fed a high-grain diet had lower levels of catalase and glutathione peroxidase (GPx) activity and total antioxidant capacity than cows fed a low-grain diet; however, super oxide dismutase (SOD) activity and malondialdehyde (MDA) levels were higher in both the liver and the plasma of high-grain than in low-grain cows. Positive correlations were observed between plasma LPS versus hepatic MDA, plasma MDA, and hepatic SOD activity, whereas hepatic GPx and plasma GPx were negatively correlated with plasma LPS. The relative mRNA abundances of GPX1 and CAT were significantly lower in the liver of cows fed a high-grain diet than those fed a low-grain diet, whereas SOD1 was significantly higher in cows fed a high-grain diet than cows fed a low-grain diet. The expression levels of Nrf2, NQO1, MT1E, UGT1A1, MGST3, and MT1A were downregulated, whereas NF-kB was upregulated, in cows fed a high-grain diet. Furthermore, nuclear factor E2-related factor 2 (Nrf2) total protein and mRNA levels were significantly lower than in low-grains. Our results demonstrate the relationship between the translocated LPS and the suppression of cellular antioxidant defense capacity, which lead to increased oxidative stress and suggests that the Nrf2-dependent antioxidant response may be affected by higher levels of LPS translocated to the bloodstream.

Differential regulation of the expression of aquaporins 3 and 9 by Auphen and dbcAMP in the SMMC-7721 hepatocellular carcinoma cell line.

Aquaglycero-aquaporins (agAQPs) are the structural foundation of rapid water transport and they appear to participate in cancer proliferation and malignancy. AQP3 expression is increased and AQP9 expression is decreased in hepatocellular carcinoma (HCC) compared to normal liver, which suggests their possible use as targets for cancer treatment. AQP-based modifiers, such as Auphen and dibutyryladenosine 3', 5'-cyclic monophosphate (dbcAMP), might be used to treat several diseases and as chemical tools for assessing the functions of AQPs in biological systems. We investigated the effects of both Auphen on AQP3 and dbcAMP on AQP9 in SMMC-7721 cells. We used western blotting, real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry to evaluate changes in AQP3 and AQP9 expression in SMMC-7721 cells after culturing with Auphen and dbcAMP, respectively. We also determined the proliferation of SMMC-7721 cells. We found that compared to HL-7702 (L02) liver cells, Auphen increased AQP3 expression in tumor cells, whereas dbcAMP decreased expression of AQP9 in these cells. Also, high concentrations of Auphen and dbcAMP inhibited proliferation of SMMC-7721 cells in vitro. Auphen and dbcAMP may inhibit HCC development and could be considered targets for HCC diagnosis and therapy.

Expression, localization and possible functions of aquaporins 3 and 8 in rat digestive system.

Although aquaporins (AQPs) play important roles in transcellular water movement, their precise quantification and localization remains controversial. We investigated expression levels and localizations of AQP3 and AQP8 and their possible functions in the rat digestive system using real-time polymerase chain reactions, western blot analysis and immunohistochemistry. We investigated the expression levels and localizations of AQP3 and AQP8 in esophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, proximal and distal colon, and liver. AQP3 was expressed in the basolateral membranes of stratified epithelia (esophagus and forestomach) and simple columnar epithelia (glandular stomach, ileum, and proximal and distal colon). Expression was particularly abundant in the esophagus, and proximal and distal colon. AQP8 was found in the subapical compartment of columnar epithelial cells of the jejunum, ileum, proximal colon and liver; the most intense staining occurred in the jejunum. Our results suggest that AQP3 and AQP8 play significant roles in intestinal function and/or fluid homeostasis and may be an important subject for future investigation of disorders that involve disruption of intestinal fluid homeostasis, such as inflammatory bowel disease and irritable bowel syndrome.

Adaption of SYBR Green-based reagent kit for real-time PCR quantitation of GC-rich DNA.

In the mammalian genome, approximately 50% of all genes are controlled by promoters with high GC contents. Analyzing the epigenetic mechanisms regulating their expression is difficult. Hence, we examined a method for stable quantification of such GC-rich DNA sequences. Quantification of DNA during real-time PCR is often based on reagent kits containing the fluorescent dye SYBR Green. However, these ready-made kits may not be suitable for amplifying DNA samples with a high GC content (>70%). DNA segments with eccentric GC contents are frequently found in proximal promoter areas, and their quantification may be necessary in chromatin accessibility by real-time polymerase chain reaction or chromatin immunoprecipitation analyses of epigenetic mechanisms of gene regulation. We therefore optimized the SYBR Green I FastStart reaction system by supplementing the system with dimethyl sulfoxide, betaine, and increased DNA polymerase content. Here, we describe the development of the assay and demonstrate its effectiveness for two different DNA templates, showing that these modifications allow for the reliable amplification and quantification of DNA with GC contents exceeding >70% using the LightCycler instrument.

Effect of high-concentrate diet on amino acid transporter expression and milk quality in Holstein dairy cows.

In order to evaluate the effect of high-concentrate diet supplementation on milk protein content, six Holstein dairy cows were assigned into high-concentrate diet (HC) or low-concentrate diet (LC) groups (N = 3/group) for 50 days. With regard to milk protein, HC feeding significantly reduced the percentage of milk protein (P < 0.01), and milk protein yield also reduced. The milk somatic cell count numbers and N-acetyl-D-glucosaminidase activity was significantly higher (P < 0.01) in the HC group than in the LC group. A pre-column derivatization procedure of o-phthalaldehyde was used to analyze the milk amino acid profile, the contents of Asp, Gln, Ala, Ile, Leu, and Lys were significantly lower in milk (P < 0.05), but Arg and Phe were significantly higher (P < 0.05) in the HC group than in the LC group. The mRNA abundance for amino acid transporters SLC7A8, SLC7A10 (P < 0.05), SLC1A3 (P < 0.05), and SLC16A10 (P < 0.05) were decreased in the HC group. These data indicate that expression of amino acid transporters alters regulation of amino acid utilization and decreases milk quality in dairy cows.

Endogenous LPS alters liver GH/IGF system gene expression and plasma lipoprotein lipase in goats.

Endotoxin lipopolysaccharide (LPS) affects the ruminant health and animal performance. The main purposes of this study were to investigate the potential effects of GH/IGF system and lipoprotein lipase (LPL) concentration on resistance the circulating LPS concentration increased in liver with high concentrate diet treatment. Non-lactating goats were randomly allocated to two groups: a high-concentrate diet (HCD) or a low-concentrate diet (LCD) in cross over design and the blood collection at different time points after feeding at the end of the experiment. The average rumen pH was significantly reduced (P<0.05), but the duration with pH was not more than 120 min in the HCD group. The plasma LPL concentration was significantly raised (P<0.05). However, from 2 h onwards, LPS concentration was significantly reduced (P<0.01) in the HCD group compared with LCD group. In addition, the plasma IGF1 concentration and the hepatic insulin-like growth factor-1 receptor (IGF1R) mRNA expression were markedly reduced (P<0.05). However, growth hormone (GH) secretion at 15, 30, and 45 min after feeding and growth hormone receptor (GHR) mRNA expression in the liver was significantly increased (P<0.05) in HCD group. The correlation analysis showed that the plasma LPL concentration was positively correlated with hepatic GHR mRNA expression (P<0.05). Conversely, the plasma LPS concentration was negatively correlated with LPL concentration (P<0.05). These findings reveal that alterations in GH/IGF system function in response to a high-concentrate diet are accompanied by corresponding changes in systemic LPL in non-lactating goats' liver in presence of endogenous LPS stress.

Identification of Differentially Expressed Proteins in Liver in Response to Subacute Ruminal Acidosis (SARA) Induced by High-concentrate Diet.

The aim of this study was to evaluate protein expression patterns of liver in response to subacute ruminal acidosis (SARA) induced by high-concentrate diet. Sixteen healthy mid-lactating goats were randomly divided into 2 groups and fed either a high-forage (HF) diet or a high-concentrate (HC) diet. The HC diet was expected to induce SARA. After ensuring the occurrence of SARA, liver samples were collected. Proteome analysis with differential in gel electrophoresis technology revealed that, 15 proteins were significantly modulated in liver in a comparison between HF and HC-fed goats. These proteins were found mainly associated with metabolism and energy transfer after identified by matrix-assisted laser desorption ionization/time of flight. The results indicated that glucose, lipid and protein catabolism could be enhanced when SARA occurred. It prompted that glucose, lipid and amine acid in the liver mainly participated in oxidation and energy supply when SARA occurred, which possibly consumed more precursors involved in milk protein and milk fat synthesis. These results suggest new candidate proteins that may contribute to a better understanding of the mechanisms that mediate liver adaptation to SARA.

High concentrate-induced subacute ruminal acidosis (SARA) increases plasma acute phase proteins (APPs) and cortisol in goats.

The aim of this study was to investigate changes of stress status in dairy goats induced to subacute ruminal acidosis (SARA). The level of acute phase proteins (APPs) including haptoglobin (HP) and serum amyloid A (SAA) in plasma and their mRNA expression in liver, as well as plasma cortisol and genes expression of key factors controlling cortisol synthesis in adrenal cortex were compared between SARA and control goats. SARA was induced by feeding high concentrate diet (60% concentrate of dry matter) for 3 weeks (SARA, n=6), while control goats (Con, n=6) received a low concentrate diet (40% concentrate of dry matter) during the experimental time. SARA goats showed ruminal pH below 5.8 for more than 3 h per day, which was significantly lower than control goats (pH>6.0). SARA goats demonstrated a significant increase of hepatic HP and SAA mRNA expression (P<0.05), and the level of HP but not SAA in plasma was markedly increased compared with control (P<0.05). The level of cortisol in plasma showed a trend to increase in SARA goats (0.050.05). These results suggested that SARA goats experienced a certain stress status, exhibiting an increase in HP production and cortisol secretion.

Resistance of Haemophilus influenzae isolates in children under 5 years old with acute respiratory infections in China between 2000 and 2002.

This prospective, three-centre study tested for antimicrobial susceptibility in 898 isolates of Haemophilus influenzae between 2000 and 2002 in Chinese children aged under 5 years with acute upper respiratory tract infection. The average incidence of beta-lactamase production was 12.0%. Overall, 88.0% of isolates were susceptible to ampicillin, 100.0% were susceptible to amoxicillin/clavulanic acid, ceftriaxone, cefuroxime and azithromycin, and 99.0% were susceptible to ciprofloxacin. Isolates from Beijing and Shanghai had a lower susceptibility to tetracycline (57.0% and 61.0%, respectively) compared with those from Guangzhou (81.0%), while trimethoprim/sulfamethoxazole susceptibilities in Shanghai (47.0%) and Guangzhou (54.0%) were significantly higher than in Beijing (35.0%). A total of 34.5% of all the isolates were susceptible to all eight of these antimicrobial agents and 12.8% were multi-drug resistant. Ampicillin resistance increased over the duration of the study. These findings show that beta-lactamase production and ampicillin resistance among isolates from Chinese children with upper respiratory tract infection are increasing, and highlight the strong correlation between ampicillin resistance and resistance to cefaclor, chloramphenicol and tetracycline in H. influenzae isolates.

Over-expression of inducible heat shock protein 70 in the gastric mucosa of partially sleep-deprived rats.

BACKGROUND: Previous findings have demonstrated increased expression of inducible heat shock protein 70 (iHSP70) in the gastric mucosa of rats exposed to partial sleep deprived (PSD). The purpose of this study was to investigate the functional role of iHSP70 and its relationship with acid secretion in the stomachs of PSD animals. METHODS: A slowly rotating drum was used to induce PSD in male Sprague-Dawley rats with or without omeprazole treatment. Gastric mucosal samples were harvested for iHSP70 mRNA and protein analysis with RT-PCR and Western blotting, respectively. Enzyme immunoassay was used to determine plasma gastrin level and gastric acidity was measured by titration. The modulating effect of PSD on 0.6 M hydrochloric acid (HCl)-induced gastric damage was also evaluated. RESULTS: PSD increased plasma gastrin, gastric acidity and expression of iHSP70, while significantly reducing HCl-induced gastric damage. Omeprazole administration decreased gastric acidity and reversed iHSP70 over-expression in PSD rats. CONCLUSIONS: PSD increases gastric acidity which enhances expression of mucosal iHSP70. Over-expression of iHSP70 may be a protective homeostatic response of the stomach to stress induced by PSD and acid secretion.

Sleep deprivation increase the expression of inducible heat shock protein 70 in rat gastric mucosa.

AIM: To investigate if sleep deprivation is able to increase the expression of inducible heat shock protein 70 in gastric mucosa and its possible role in mucosal defense. METHODS: Rats for sleep disruption were placed inside a computerized rotating drum, gastric mucosa was taken from rats with 1, 3 and 7d sleep deprivation. RT-PCR, immunohistochemistry and Western blotting were used to determine the expression of heat shock protein 70. Ethanol (500mL.L(-1), i.g.) was used to induce gastric mucosa damage. RESULTS: RT-PCR, Western blotting and immunostaining confirmed that the sleep deprivation as a stress resulted in significantly greater expression of inducible heat shock protein 70 in gastric mucosa of rats. After the 500mL.L(-1) ethanol challenge, the ulcer area found in the rats with 7d sleep deprivation (19.15 +/- 4.2)mm(2) was significantly lower (P<0.01) than the corresponding control (53.7 +/- 8.1) mm(2). CONCLUSION: Sleep deprivation as a stress, in addition to lowering the gastric mucosal barrier, is able to stimulate the expression of inducible heat shock protein 70 in gastric mucosa of rats, the heat shock protein 70 may play an important role in gastric mucosal protection.

Development of Female Contraceptive Vaccine Through DNA Inoculation of Human Chorionic Gonadotropin Beta Subunit (hCGß).

Human chorionic gonadotropin (hCG) has been considered as a primary target molecule for a contraceptive vaccine by the World Health Organization because of its physiological and temporal specificity. hCG is an essential factor for the successful implantation and establishment of early pregnancy. For a decade, the most advanced works in development of a contraceptive vaccine have been concentrated on polypeptide based vaccines targeted directly against hCG. Although many studies have reached the stage of clinical testing using a number of prototype vaccines based on different parts of hCG molecule, their efficacy has been disappointing (1-4).

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS-1 proteins.

Human gastric cancer MKN-45 cells were transfected with pULB 3238, a plasmid carrying MVMp NS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectants died, while others remained alive, but the growth features of survived cells were changed. For further study on the antineoplastic function of parvoviral NS-1 protein in vivo, transgenic mice carrying NS-1 genes were established by conventional method. Among 4 founders, one of them was found to be able to transmit the transgene to around 50% of their offsprings. RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

[Effect of berberine on transit time of human small intestine].

Sorbitol was used as a test sugar for the determination of small intestinal transit time (SITT) by means of breath hydrogen test (BHT). After oral administration of 15g sorbitol, breath hydrogen increased markedly (delta H2 > 5 mumol/L) in 26 of 30 subjects. Following ingestion of a mixture of meglucamine diatrizoate and sorbitol by 18 subjects, SITT measured by BHT correlated closely with the simultaneously determined time for the meglucamine diatrizoate in reaching ileo-cecum. The BHT was used to investigate the effect of berberine on SITT in human. SITT in 20 healthy subjects was 71.10 +/- 22.04 min, SITT was significantly delayed to 98.25 +/- 29.03 min after oral administration of the 1.2g of berberine (P < 0.01). This result suggested that the antidiarrheal property of berberine might be mediated, at least in part, by its ability to delay the small intestinal transit.