CD47-SIRPα Blockade Sensitizes Head and Neck Squamous Cell Carcinoma to Cetuximab by Enhancing Macrophage Adhesion to Cancer Cells.

Developing effective treatments for patients with head and neck squamous cell carcinoma (HNSCC) is a significant challenge. Cetuximab, a first-line targeted therapy for HNSCC, exhibits limited efficacy. Here, we used pooled CRISPR screening to find targets that can synergize with cetuximab and identified CD47 as the leading candidate. Rather than inhibiting cancer cell proliferation, CD47 inhibition promoted cetuximab-triggered antibody-dependent cellular phagocytosis (ADCP), thereby enhancing macrophage-mediated cancer cell removal. The combination of CD47-SIRPα blockade and cetuximab demonstrated strong anticancer activity in vivo. In addition to blocking the phagocytosis checkpoint, CD47-SIRPα inhibition upregulated CD11b/CD18 on the surface of macrophages, which accelerated intercellular adhesion between macrophages and cancer cells to enhance subsequent phagocytosis. Inhibition of the interaction between macrophage CD11b/CD18 and cancer cell ICAM1 eliminated the intercellular adhesion and phagocytosis induced by CD47-SIRPα blockade. Thus, CD47-SIRPα blockade enhances ADCP through CD11b/CD18-ICAM1-mediated intercellular adhesion and sensitizes HNSCC to cetuximab.

Clinical and Functional Studies of MTOR Variants in Smith-Kingsmore Syndrome Reveal Deficits of Circadian Rhythm and Sleep-Wake Behavior.

Heterozygous de novo or inherited gain-of-function mutations in the MTOR gene cause Smith-Kingsmore syndrome (SKS). SKS is a rare autosomal dominant condition, and individuals with SKS display macrocephaly/megalencephaly, developmental delay, intellectual disability, and seizures. A few dozen individuals are reported in the literature. Here, we report a cohort of 28 individuals with SKS that represent 9 MTOR pathogenic variants. We conducted a detailed natural history study and found pathophysiological deficits among individuals with SKS, in addition to the common neurodevelopmental symptoms. These symptoms include sleep-wake disturbance, hyperphagia, and hyperactivity, indicative of homeostatic imbalance. To characterize these variants, we developed cell models and characterized their functional consequences. We showed that these SKS variants display a range of mTOR activities and respond to the mTOR inhibitor, rapamycin, differently. For example, the R1480_C1483del variant we identified here and the previously known C1483F are more active than wild-type controls and less responsive to rapamycin. Further, we showed that SKS mutations dampened circadian rhythms and low-dose rapamycin improved the rhythm amplitude, suggesting that optimal mTOR activity is required for normal circadian function. As SKS is caused by gain of function mutations in MTOR, rapamycin was used to treat several patients. While higher doses of rapamycin caused delayed sleep-wake phase disorder in a subset of patients, optimized lower doses improved sleep. Our study expands the clinical and molecular spectrum of SKS and support further studies for mechanism-guided treatment options to improve sleep-wake behavior and overall health.

The interaction between innate immunity and oral microbiota in oral diseases.

INTRODUCTION: Innate immunity serves as the frontline to combat invading pathogens. Oral microbiota is the total collection of microorganisms colonized within the oral cavity. By recognizing the resident microorganisms through pattern recognition receptors, innate immunity is capable of interacting with oral microbiota and maintaining homeostasis. Dysregulation of interaction may lead to the pathogenesis of several oral diseases. Decoding the crosstalk between oral microbiota and innate immunity may be contributory to developing novel therapies for preventing and treating oral diseases. AREAS COVERED: This article reviewed pattern recognition receptors in the recognition of oral microbiota, the reciprocal interaction between innate immunity and oral microbiota, and discussed how the dysregulation of this relationship leads to the pathogenesis and development of oral diseases. EXPERT OPINION: Many studies have been conducted to illustrate the relationship between oral microbiota and innate immunity and its role in the occurrence of different oral diseases. The impact and mechanisms of innate immune cells on oral microbiota and the mechanisms of dysbiotic microbiota in altering innate immunity are still needed to be investigated. Altering the oral microbiota might be a possible solution for treating and preventing oral diseases.

Effects of biochar and crop straws on the bioavailability of cadmium in contaminated soil.

Numerous studies have been investigated the potential of biochar (BC) derived from various materials and crop straw (CS) to decrease the bioavailability of heavy metals in soil contaminated with cadmium (Cd), and thereby reduce their potential risk to human health and the ecological environment. However, little attention has been given to the comparison of heavy metal remediation efficiency using BC and CS such as peanut vine (PV) and rice straw (RS), especially in soil contaminated with Cd. Here, we explore if Cd bioavailability is affected in contaminated soil by BC and CS. Peanuts were grown in plastic pots, which contained BC or CS at 5% (dry weight, w/w) in controlled environment mesocosms. The bioavailability of Cd in contaminated soil was measured by Cd concentration in the plant and the concentrations of various forms of Cd in the soil. At the same plant age, growth with BC (compared with PV and RS) led to 13.56% and 8.28% lower rates of Cd content in the aboveground parts, 40.65% and 35.67% lower rates of Cd content in the seeds, yet 9.08% and 7.09% lower rates of Cd content in the roots, yet 35.80% and 28.48% lower rates of exchangeable Cd content in the soil. Moreover, BC amendment enhanced the biomass of peanut and physiological quality. Thus, BC had a greater impact on immobilizing Cd in the soil. The results imply that BC was more significantly (P < 0.05) remarkable in decreasing the Cd bioavailability and improving the biomass of peanut. BC has greater potential for enhancing soil quality and promoting peanut growth. In conclusion, this research demonstrates an understanding of employing BC as a promising inexpensive and eco-friendly amendment to remediate soil contaminated with Cd.

Evaluation of filamentous heterocystous cyanobacteria for integrated pig-farm biogas slurry treatment and bioenergy production.

The study evaluates 36 filamentous heterocystous cyanobacteria for the treatment of biogas slurry from pig farm and the accumulation of biomass for bioenergy production. The results showed that only the strains B, J, and L were able to adapt to a 10% biogas slurry. The removal rates of ammonia nitrogen, total nitrogen, and total phosphorus for strains J and L were 92.46%-97.97%, 73.79%-79.90%, and 97.14%-98.46%, respectively, higher than that of strain B. Strain J had the highest biomass productivity and lipid productivity. Based on the biodiesel prediction results, it was concluded that strains J and L are more suitable for biodiesel production. The estimation of theoretical methane potential suggests that the algal biomass of strain J also have the desirable possibility of biogas generation. In summary, algal strain J (Nostoc sp.) offers great potential for biogas slurry treatment and for the production of bioenergy.

The compound effects of biochar and iron on watercress in a Cd/Pb-contaminated soil.

Pot experiment was conducted to evaluate the effect of two types of biochar (2% (w/w)), Pennisetum sinese Roxb biochar (PB) and coffee grounds biochar (CB), combined with iron fertilizer (1.3 g kg-1 Fe) on the growth, quality, Cd/Pb accumulation in watercress, soil physicochemical properties, soil fertility, soil enzyme activities, and fraction distribution of Cd/Pb in soil. The results showed that the two types of biochar combined with iron fertilizer (BC-Fe) amendments could increase the shoot height, root length, plant biomass, soluble sugar and soluble protein of watercress, soil pH value, soil organic matter (SOM), ammonium nitrogen (NH4+-N), available phosphorus, and available potassium. CB-Fe amendment enhanced soil urease, sucrose, and catalase activities, while PB-Fe amendment only enhanced soil urease activity among the three enzymes. The two BC-Fe amendments decreased exchangeable-Cd/Pb and reducible-Cd/Pb concentrations, while enhanced oxidizable-Cd/Pb and residual-Cd/Pb concentrations. Furthermore, the two BC-Fe amendments decreased significantly Cd and Pb accumulation in watercress root and shoot. The reduction rate for Cd and Pb in shoot by 42.9%, 20.0%, and 68.2%, 58.4% under PB-Fe and by 38.1%, 20%, and 62.5%, 48.8% under CB-Fe, respectively, for the first crop and the second crop. In conclusion, BC-Fe amendment could improve soil physicochemical properties and soil fertility, promote Cd and Pb transfer to the stable form, thus, reduce the bioavailability and mobility of Cd and Pb, and further, decrease Cd and Pb ecotoxicity and its accumulation in watercress and improve watercress quality.

YY1's role in the Peg3 imprinted domain.

The ICR (Imprinting Control Region) of the Peg3 (Paternally Expressed Gene 3) domain contains an unusual cluster of YY1 binding sites. In the current study, these YY1 binding sites were mutated to characterize the unknown roles in the mouse Peg3 domain. According to the results, paternal and maternal transmission of the mutant allele did not cause any major effect on the survival of the pups. In the mutants, the maternal-specific DNA methylation on the ICR was properly established and maintained, causing no major effect on the imprinting of the domain. In contrast, the paternal transmission resulted in changes in the expression levels of several genes: down-regulation of Peg3 and Usp29 and up-regulation of Zim1. These changes were more pronounced during the neonatal stage than during the adult stage. In the case of Peg3 and Zim1, the levels of the observed changes were also different between males and females, suggesting the different degrees of YY1 involvement between two sexes. Overall, the results indicated that YY1 is mainly involved in controlling the transcriptional levels, but not the DNA methylation, of the Peg3 domain.

Inversion of the imprinting control region of the Peg3 domain.

The imprinting of the mouse Peg3 domain is controlled through a 4-kb genomic region encompassing the bidirectional promoter and 1st exons of Peg3 and Usp29. In the current study, this ICR was inverted to test its orientation dependency for the transcriptional and imprinting control of the Peg3 domain. The inversion resulted in the exchange of promoters and 1st exons between Peg3 and Usp29. Paternal transmission of this inversion caused 10-fold down-regulation of Peg3 and 2-fold up-regulation of Usp29 in neonatal heads, consistent with its original promoter strength in each direction. The paternal transmission also resulted in reduced body size among the animals, which was likely contributed by the dramatic down-regulation of Peg3. Transmission through either allele caused no changes in the DNA methylation and imprinting status of the Peg3 domain except that Zfp264 became bi-allelic through the maternal transmission. Overall, the current study suggests that the orientation of the Peg3-ICR may play no role in its allele-specific DNA methylation, but very critical for the transcriptional regulation of the entire imprinted domain.

Parental and sexual conflicts over the Peg3 imprinted domain.

In the current study, the imprinting control region of the mouse Peg3 domain was deleted to test its functional impact on animal growth and survival. The paternal transmission of the deletion resulted in complete abolition of the transcription of two paternally expressed genes, Peg3 and Usp29, causing the reduced body weight of the pups. In contrast, the maternal transmission resulted in the unexpected transcriptional up-regulation of the remaining paternal allele of both Peg3 and Usp29, causing the increased body weight and survival rates. Thus, the imprinted maternal allele of the ICR may be a suppressor antagonistic to the active paternal allele of the ICR, suggesting a potential intralocus allelic conflict. The opposite outcomes between the two transmissions also justify the functional compromise that the maternal allele has become epigenetically repressed rather than genetically deleted during mammalian evolution. The mice homozygous for the deletion develop normally but with a skewed sex ratio, one male per litter, revealing its sex-biased effect. Overall, the Peg3 locus may have evolved to an imprinted domain to cope with both parental and sexual conflicts driven by its growth-stimulating paternal versus growth-suppressing maternal alleles.

PEG3 Interacts with KAP1 through KRAB-A.

Peg3 (Paternally Expressed Gene 3) is an imprinted gene that encodes a zinc finger DNA-binding protein. Peg3 itself is localized in the middle of a KRAB-A (Kruppel-Associated Box) zinc finger gene cluster. The amino acid sequence encoded by its exon 7 also shows sequence similarity to that of KRAB-A, suggesting Peg3 as a KRAB-containing zinc finger gene. As predicted, the PEG3 protein was co-immunoprecipitated with KAP1, a co-repressor that interacts with KRAB-A. A series of follow-up experiments further demonstrated that the exon 7 of PEG3 is indeed responsible for its physical interaction with KAP1. ChIP and promoter assays also indicated that PEG3 likely controls its downstream genes through the KAP1-mediated repression mechanism. Overall, the current study identifies PEG3 as a KRAB-containing zinc finger protein that interacts with the co-repressor protein KAP1.

PEG3 binds to H19-ICR as a transcriptional repressor.

Paternally expressed gene 3 (Peg3) encodes a DNA-binding protein with 12 C2H2 zinc finger motifs. In the current study, we performed ChIP-seq using mouse embryonic fibroblast (MEF) cells. This experiment identified a set of 16 PEG3 genomic targets, the majority of which overlapped with the promoter regions of genes with oocyte expression. These potential downstream genes were upregulated in MEF cells lacking PEG3 protein, suggesting a potential repressor role for PEG3. Our study also identified the imprinting control region (ICR) of H19 as a genomic target. According to the results, PEG3 binds to a specific sequence motif located between the 3rd and 4th CTCF binding sites of the H19-ICR. PEG3 also binds to the active maternal allele of the H19-ICR. The expression levels of H19 were upregulated in MEF cells lacking PEG3, and this upregulation was mainly derived from the maternal allele. This suggests that PEG3 may function as a transcriptional repressor for the maternal allele of H19. Overall, the current study uncovers a potential functional relationship between Peg3 and H19, and also confirms PEG3 as a transcriptional repressor for the identified downstream genes.

Transcriptional Truncation of the Long Coding Imprinted Gene Usp29.

Usp29 (Ubiquitin-specific protease 29) is a paternally expressed gene located upstream of another imprinted gene Peg3. In the current study, the transcription of this long coding gene spanning a 250-kb genomic distance was truncated using a knockin allele. According to the results, paternal transmission of the mutant allele resulted in reduced body and litter sizes whereas the maternal transmission caused no obvious effects. In the paternal mutant, the expression levels of Usp29 were reduced to 14-18% level of the wild-type littermates due to the Poly-A signal included in the knockin cassette. Expression analyses further revealed an unusual female-specific up-regulation of the adjacent imprinted gene Zfp264 in the mutant. Consistent with this, the promoter of Zfp264 was hypomethylated only in the female mutant. Interestingly, this female-specific hypomethylation by the knockin allele was not detected in the offspring of an interspecific crossing, indicating its sensitivity to genetic background. Overall, the results suggest that the transcription of Usp29 may be involved in DNA methylation setting of Zfp264 promoter in a sex-specific manner.

Effects of perchlorate bioaccumulation on Spodoptera litura growth and sex ratio.

Perchlorate (ClO4 (-)) pollution is widespread in the natural environment, but the effects of ClO4 (-) on the terrestrial insects are rarely studied. Here, when phytophagous insect Spodoptera litura larvae were fed on the diets with different ClO4 (-) concentrations, changes in their life-history traits were recorded; ClO4 (-) accumulations in feces and insect body were detected. The results demonstrated that ClO4 (-) bioaccumulation in insect at the different developmental stages was ranked in the order: adults > pupae > the 4th > 5th > 6th instar larvae. Besides, ClO4 (-) accumulations in the feces were ranked in the order: the 6th > 5th > 4th instar larvae. The ClO4 (-) accumulations in female pupae and adults were significantly higher than that in males. ClO4 (-) bioaccumulation in insect prolonged larval development time and caused a skewed sex ratio (the percentage of males at metamorphosis significantly decreased) under 100 to 200 mg ClO4 (-)/kg treatment. Therefore, ClO4 (-) accumulations in S. litura body presented developmental stage-, sex-specific pattern, and the sex-specific ClO4 (-) accumulations resulted in difference of sex ratio. These effects were observed at concentrations reported in natural environments contaminated with ClO4 (-), suggesting that this contaminant may pose a threat to the normal development and growth of this insect species.

Varietal differences in the growth of rice seedlings exposed to perchlorate and their antioxidative defense mechanisms.

A hydroponic experiment was conducted to investigate perchlorate (ClO4 (-) ) phytotoxicity in different rice varieties. Considerable variations were observed when 24 rice varieties were treated with ClO4 (-) . The shoot height, root length, and biomass of most varieties were significantly reduced by ClO4 (-) . The roots were more sensitive than the shoots. Hierarchical clustering analysis demonstrated primarily 4 groups: ClO4 (-) -sensitive, medium ClO4 (-) -sensitive, medium ClO4 (-) tolerant, and ClO4 (-) -tolerant. Gannuoxiang (a ClO4 (-) -tolerant variety) and IR65598-112-2 (a ClO4 (-) -sensitive variety) were chosen to explore their antioxidant response when exposed to 0.2 mmol/L, 2.0 mmol/L, and 4.0 mmol/L ClO4 (-) . The results showed that the activities of superoxide dismutase and catalase increased in the shoots and roots of gannuoxiang with increasing doses of ClO4 (-) , but both of them decreased at higher concentrations of ClO4 (-) in IR65598-112-2. The addition of ClO4 (-) led to a significant increase in peroxidase activities for both of the varieties, whereas the increase was more pronounced in gannuoxiang than in IR65598-112-2. No significant difference was found in malondialdehyde (MDA) contents in gannuoxiang, whereas the addition of ClO4 (-) increased the MDA level significantly in IR65598-112-2. The results indicated that gannuoxiang has higher activities of antioxidant enzymes than IR65598-112-2 to cope with oxidative damage caused by ClO4 (-) stress, which may be the main cause of its high tolerance.

The role of dentin matrix protein 1 (DMP1) in regulation of osteogenic differentiation of rat dental follicle stem cells (DFSCs).

OBJECTIVES: Primary isolated dental follicle stem cells (DFSCs) possess a strong osteogenesis capability, and such capability is reduced during in vitro culture. Because dentin matrix protein 1 (DMP1) is essential in the maturation of osteoblasts, our objectives were to determine (1) the expression of DMP1 in the DFSCs, (2) the correlation between DMP1 expression and osteogenic capability of DFSCs, and (3) the ability of DMP1 to promote osteogenic differentiation of DFSCs. METHODS: DFSCs and their non-stem cell counterpart dental follicle cells (DFC) were established from postnatal rat pups. Expression of DMP1 in the DFSCs and DFC was determined using real-time RT-PCR and western blotting. Different passages of DFSCs were subjected to osteogenic induction. The correlation between osteogenesis and DMP1 expression was analyzed. Then, expression of DMP1 in the DFSCs was knocked-down using siRNA, followed by osteogenic induction to evaluate the effect of DMP1-knockdown. Finally, the late passage DFSCs with reduced DMP1 expression and osteogenic capability were cultured in osteogenic induction medium containing mouse recombinant DMP1 (mrDMP1) to determine if DMP1 can restore osteogenesis of DFSCs. RESULTS: DFSCs expressed much higher levels of DMP1 than did DFC. DMP1 expression was correlated with the osteogenic capability of DFSCs. Knockdown of DMP1 expression markedly decreased the osteogenesis and osteogenic gene expression in the DFSCs whereas adding mrDMP1 protein to the osteogenic induction medium enhanced osteogenesis. CONCLUSIONS: DMP1 is highly expressed in the DFSCs, but minimally expressed in non-stem cell DFC. DMP1 appears to play an important role for osteogenic differentiation of the DFSCs.

Regulation and function of the peg3 imprinted domain.

A subset of mammalian genes differ functionally between two alleles due to genomic imprinting, and seven such genes (Peg3, Usp29, APeg3, Zfp264, Zim1, Zim2, Zim3) are localized within the 500-kb genomic interval of the human and mouse genomes, constituting the Peg3 imprinted domain. This Peg3 domain shares several features with the other imprinted domains, including an evolutionarily conserved domain structure, along with transcriptional co-regulation through shared cis regulatory elements, as well as functional roles in controlling fetal growth rates and maternal-caring behaviors. The Peg3 domain also displays some unique features, including YY1-mediated regulation of transcription and imprinting; conversion and adaptation of several protein-coding members as ncRNA genes during evolution; and its close connection to human cancers through the potential tumor suppressor functions of Peg3 and Usp29. In this review, we summarize and discuss these features of the Peg3 domain.

Paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor.

The expression of two adjacent imprinted genes, Peg3 and Zim1, is inversely correlated: down-regulation of Peg3 coinciding with up-regulation of Zim1. The current study characterized this inverse correlation using a mutant allele targeting Peg3. According to the results, the mutation on the paternal allele of Peg3 resulted in a dramatic increase in the transcription levels of the maternal allele of Zim1, suggesting the involvement of unknown trans factors in this trans-allelic event. Subsequent ChIP experiments revealed that the protein encoded by Peg3 itself binds to the zinc finger exon of Zim1, which is modified with the repression mark H3K9me3. Interestingly, the levels of H3K9me3 on Zim1 are also reduced in the mutant cells lacking the protein PEG3, suggesting potential roles for PEG3 in establishing H3K9me3 on Zim1. Reintroducing PEG3 into the mutant cell restored down-regulation of Zim1, confirming the predicted repressor role for Peg3 on Zim1. Overall, these results demonstrated that paternally expressed Peg3 controls maternally expressed Zim1 as a trans factor. The current study also provides the first case for the trans-allelic interaction of two oppositely imprinted genes through their gene products.

Identification of tumor antigens that elicit a humoral immune response in the sera of Chinese esophageal squamous cell carcinoma patients by modified serological proteome analysis.

Our aim was to identify novel tumor-associated antigens from the esophageal squamous cell carcinoma (ESCC) cell line EC0156, and related autoantibodies in sera from patients with ESCC. We used modified serological proteome analysis, involving one- and two-dimensional electrophoresis, Western blot, and MALDI-TOF/TOF-MS to identify 6 ESCC-associated antigens. From these, 105 kDa heat shock protein (HSP105) and triosephosphate isomerase (TIM) were further evaluated and we determined they could induce autoantibody responses in ESCC sera and are highly expressed in ESCC tissues. Anti-HSP105 and anti-TIM autoantibodies were found in 39.1% (18/46) and 34.8% (16/46) of patients with ESCC, respectively, but only in two controls. A receiver operating characteristic curve constructed with HSP105 and TIM gave a sensitivity of 54.3% and 95% (38/40) specificity in discriminating ESCC from matched controls. Interestingly, we found that autoantibodies against TIM in ESCC serum mainly reacted with glycosylated but not deglycosylated TIM. The preliminary results suggest the potential utility of screening autoantibodies in sera for use as biomarkers for cancer diagnosis.

Effects of perchlorate on growth of four wetland plants and its accumulation in plant tissues.

Perchlorate contamination in water is of concern because of uncertainties about toxicity and health effects, impact on ecosystems, and possible indirect exposure pathways to humans. Therefore, it is very important to investigate the ecotoxicology of perchlorate and to screen plant species for phytoremediation. Effects of perchlorate (20, 200, and 500 mg/L) on the growth of four wetland plants (Eichhornia crassipes, Acorus calamus L., Thalia dealbata, and Canna indica) as well as its accumulation in different plant tissues were investigated through water culture experiments. Twenty milligrams per liter of perchlorate had no significant effects on height, root length, aboveground part weight, root weight, and oxidizing power of roots of four plants, except A. calamus, and increasing concentrations of perchlorate showed that out of the four wetland plants, only A. calamus had a significant (p<0.05) dose-dependent decrease in these parameters. When treated with 500 mg/L perchlorate, these parameters and chlorophyll content in the leaf of plants showed significant decline contrasted to control groups, except the root length of E. crassipes and C. indica. The order of inhibition rates of perchlorate on root length, aboveground part weight and root weight, and oxidizing power of roots was: A. calamus > C. indica > T. dealbata > E. crassipes and on chlorophyll content in the leaf it was: A. calamus > T. dealbata > C. indica > E. crassipes. The higher the concentration of perchlorate used, the higher the amount of perchlorate accumulation in plants. Perchlorate accumulation in aboveground tissues was much higher than that in underground tissues and leaf was the main tissue for perchlorate accumulation. The order of perchlorate accumulation content and the bioconcentration factor in leaf of four plants was: E. crassipes > C. indica > T. dealbata > A. calamus. Therefore, E. crassipes might be an ideal plant with high tolerance ability and accumulation ability for constructing wetland to remediate high levels of perchlorate polluted water.

Individual and joint toxicity of three chloroacetanilide herbicides to freshwater cladoceran Daphnia carinata.

Individual and joint toxicity of three chloroacetanilide herbicides to a freshwater cladoceran were studied. The 48 h-LC50 values of alachlor, acetochlor and butachlor to Daphnia carinata Dc42 were 11.1, 11.8 and 3.45 mg L(-1), respectively. The toxicity was significantly (p < 0.05) related to hydrophobicity. The additive indexes of binary mixtures of three herbicides were less than zero and it showed antagonism. The body length of D. carinata treated with high concentration of herbicides was shorter than that of control group significantly (p < 0.05). It suggests that joint actions must be considered when assessing the acute toxicity of chloroacetanilide herbicides to D. carinata.