Single-cell RNA Sequencing Analysis Reveals the Role of Cancer-associated Fibroblasts in Skin Melanoma.

AIMS: Mechanism of fibroblasts in skin melanoma (SKME) revealed by single-cell RNA sequencing data. BACKGROUND: SKME is responsible for more than 80% of skin-related cancer deaths. Cancer-associated fibroblasts (CAFs) generate inflammatory factors, growth factors and extracellular matrix proteins to facilitate cancer cell growth, metastasis, drug resistance and immune exclusion. However, molecular mechanisms of CAFs in SKME are still lacking. OBJECTIVE: Our goal was to reveal the role of CAFs in SKME. METHODS: We downloaded the single-cell RNA sequencing (scRNA-seq) dataset from the Gene Expression Omnibus (GSE215120) database. Then, the Seurat package was applied to analyze the single-cell atlas of SKME data, and cell subsets were annotated with the CellMarker database. The molecular mechanisms of CAFs in SKME were disclosed via differential gene expression and enrichment analysis, Cellchat and SCENIC methods. RESULTS: Using scRNA-seq data, three SKME cases were used and downscaled and clustered to identify 11 cell subgroups and 5 CAF subsets. The enrichment of highly expressed genes among the 5 CAF subsets suggests that cell migration-inducing hyaluronan-binding protein (CEMIP) + fibroblasts and naked cuticle homolog 1 (NKD1) + fibroblasts were closely associated with epithelial to mesenchymal transition. Cellchat analysis revealed that CAF subpopulations promoted melanocyte proliferation through Jagged1 (JAG1)-Notch homolog 1 (NOTCH1), JAG1-NOTCH3 and migration through pleiotrophin (PTN)-syndecan-3 (SDC3) receptor-ligand pairs. The SCENIC analysis identified that most of the transcription factors in each CAF subpopulation played a certain role in the metastasis of melanoma and were highly expressed in metastatic SKME samples. Specifically, we observed that CEMIP+ fibroblasts and NKD1+ fibroblasts had potential roles in participating in immune therapy resistance. Collectively, we uncovered a single-- cell atlas of SKME and revealed the molecular mechanisms of CAFs in SKME development, providing a base for immune therapy and prognosis assessment. CONCLUSION: Our study reveals that 5 CAFs in SKME have a promoting effect on melanocyte proliferation and metastasis. More importantly, CEMIP+ fibroblasts and NKD1+ fibroblasts displayed close connections with immune therapy resistance. These findings help provide a good basis for future immune therapy and prognosis assessment targeting CAFs in SKME.

A Wound Treatment Strategy for "Super Long-Term Difficult-to-Heal Wounds": A Single-Center Retrospective Study.

Chronic wounds are a challenge for clinicians. Treating chronic wounds in elderly patients is difficult due to comorbidities and poor immunity, tissue renewal, and regeneration. This study shared the therapeutic experiences of 40 patients with super long-term difficult-to-heal wounds and to describe the effects of negative pressure wound therapy (NPWT)-assisted debridement and autologous scalp grafting. Elderly patients with chronic wounds for more than 60 years who underwent NPWT-assisted debridement and autologous scalp grafting between 2015 and 2017 were retrospectively analyzed. Forty patients were identified and analyzed. Among all patients, the average wound area was 56 (interquartile range 30-90) cm2. The wound infection rate was 82.1%, and that before the first autologous scalp grafting was 51.3%. The average total number of surgeries was 3, and the number of times the NPWT device was replaced was once. A total of 97.4% of patients had one autologous scalp grafting performed. The transplanted scalp survived completely in 97.4% of patients. One hundred percent of patients had no postoperative complications and healed. The average wound healing time was 34.5 ± 10.1 days. This study showed that NPWT-assisted debridement and autologous scalp grafting have the advantages of high survival rate of the skin and decreased wound recurrence and may be a suitable treatment for super long-term difficult-to-heal wounds in elderly patients.

Effect of ammonium acetate on alcohol fermentation in cassava-alcohol fermentation process.

The cassava-alcohol fermentation process employing cassava requires nitrogen source to maximize yields by a commercial strain of S. cerevisiae TG1348. In this study, a factorial experimental design was used to assess a suitable nitrogen source for growth and fermentative performance of S. cerevisiae in cassava-ethanol fermentation. The alcohol fermentation time was about 39 h for urea and ammonium acetate, which was 48 h for ammonium chloride and ammonium sulphate. The fermentation time was reduced by 19 % when using urea and ammonium acetate as nitrogen source. Ammonium acetate leaded to the highest alcohol yield, which was 4% higher than for ammonium sulphate. In addition, byproduct formation differed obviously between the nitrogen sources. The glycerol yields were similar for urea, ammonium sulphate and ammonium chloride but were 24 % lower for ammonium acetate. However, glycerol yield for ammonium carbonate was higher than for other nitrogen sources. Clearly, in batch cultures the ammonium acetate not only increased ethanol generation, but also decreased glycerol generation. In order to understand why ammonium acetate promotes alcohol fermentation, acetic acid was added to different nitrogen sources. The weight loss effect of ammonium sulphate adding acetic acid and ammonium acetate as nitrogen source was the same. The fermentation time was shortened by adding acetic acid. And pH was increased by addition of acetic acid when ammonium sulfate and urea were used as nitrogen sources. The results showed that the acetate root plays an important role in ammonium acetate. The results of this study could facilitate the development of new strategies to control fermentation performance.

Tandem PCR: a novel and efficient unit amplification model for the preparation of small DNA fragments.

The present DNA marker preparation with PCR amplification, one primer pair for one target DNA fragment, was very tedious and labor intensive. To develop a simple and efficient system for the preparation of small DNA fragments, a novel PCR amplification pattern was designed and tested, of which targeted small DNA fragments were amplified in groups as a unit with a specific synthetic vector as template DNA. The amplified units can be different dependent on the identities of the employed primers and give out variable combinations of small DNA fragments through complete or partial restrictive digestion with EcoRI. The novel pattern made the PCR amplification of small DNA fragments not only more efficient but also more economic than ever before. The tandem PCR pattern, as the most efficient and high throughput method for small DNA fragment preparation, has wide application for the production of various DNA markers and a good complementation to the larger DNA fragment preparation by complex synthetic vector fermentation.

Unit cloning and amplification as novel and universal strategies for complex vector construction and small DNA fragment preparation.

With a novel and universal strategy for the cloning of multiple DNA fragments, a complex synthetic vector (pVEC100), harboring the target DNA fragments in conventional 100-bp DNA ladder, was constructed for efficient and large-scale production of 100-bp DNA marker through bacteria fermentation, plasmid extraction and restrictive digestion. Since the restrictive digestion of complex vectors yields insufficient small DNA fragments, an innovative PCR model was developed as an alternative. The PCR model comprised a specially designed template vector and a unit amplification model for producing groups of small DNA fragments. The unit amplification model improved the efficiency of the PCR protocol and made it more economical and easier for small DNA fragment amplification. The approach presented in this paper--a unit cloning model for constructing complex synthetic vectors combined with the modular design of unit amplification by PCR--is a powerful method for preparing small DNA fragments of DNA molecular weight standards.

Novel strategies to construct complex synthetic vectors to produce DNA molecular weight standards.

DNA molecular weight standards (DNA markers, nucleic acid ladders) are commonly used in molecular biology laboratories as references to estimate the size of various DNA samples in electrophoresis process. One method of DNA marker production is digestion of synthetic vectors harboring multiple DNA fragments of known sizes by restriction enzymes. In this article, we described three novel strategies-sequential DNA fragment ligation, screening of ligation products by polymerase chain reaction (PCR) with end primers, and "small fragment accumulation"-for constructing complex synthetic vectors and minimizing the mass differences between DNA fragments produced from restrictive digestion of synthetic vectors. The strategy could be applied to construct various complex synthetic vectors to produce any type of low-range DNA markers, usually available commercially. In addition, the strategy is useful for single-step ligation of multiple DNA fragments for construction of complex synthetic vectors and other applications in molecular biology field.

Identification and characterization of a novel adenine phosphoribosyltransferase gene (ZmAPT2) from maize (Zea mays L.).

Adenine phosphoribosyltransferase (APRT) is the key enzyme that converts adenine to adenosine monophosphate (AMP) in the purine salvage pathway. It was found that several different forms of APRT gene exist in plants, but no APRT gene in maize has been reported up to now. In this study, a novel maize APRT gene was cloned and characterized through a combination of bioinformatic, RT-PCR and RACE strategies. The full length of APRT cDNA sequence is 1202 nucleotides, with an ORF encoding 214 amino acid residues. Alignment of the deduced protein with that of other plant APRT genes indicates that the new gene is the form 2 of maize APRT, thus it was named ZmAPT2. Through basic local alignment search tool, search in the genomic survey sequence database of MaizeGDB, the putative genomic sequence of ZmAPT2 was obtained. Comparison of the cDNA and genomic sequence of the ZmAPT2 gene revealed that it contained seven exons and six introns. The locations of the introns within the maize ZmAPT2 coding region were consistent with those in the previously isolated APRTs of arabidopsis and rice. RT-PCR analysis showed that ZmAPRT was constitutively expressing in different organs under high temperature and salt stresses. Southern blot analysis indicated that at least three APRT genes existed in maize genome. These results confirmed that the novel maize ZmAPT2 gene was truly identified, and its potential role in maize growth and development was discussed.

Cloning, characterization, and transformation of the phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) in maize (Zea mays L.).

N-methylation of phosphoethanolamine, the committing step in choline (Cho) biosynthesis in plants, is catalyzed by S-adenosyl-L-methionine: phosphoethanolamine N-methyltransferase (PEAMT, EC 2.1.1.103). Herein we report the cloning and characterization of the novel maize phosphoethanolamine N-methyltransferase gene (ZmPEAMT1) using a combination of bioinformatics and a PCR-based allele mining strategy. The cDNA sequence of ZmPEAMT1 gene is 1,806 bp in length and translates a 495 amino acids peptide. The upstream promoter sequence of ZmPEAMT1 were obtained by TAIL-PCR, and contained four kinds of putative cis-acting regulatory elements, including stress-responsive elements, phytohormone-responsive elements, pollen developmental special activation elements, and light-induced signal transduction elements, as well as several other structural features in common with the promoter of rice and Arabidopsis homologies. RT-PCR analysis showed that expression of ZmPEAMT1 was induced by salt stress and suppressed by high temperature. Over-expression of ZmPEAMT1 enhanced the salt tolerance, root length, and silique number in transgenic Arabidopsis. These data indicated that ZmPEAMT1 maybe involved in maize root development and stress resistance, and maybe having a potential application in maize genetic engineering.

Management of a Rare Case With Severe Hydrofluoric Acid Burns: Important Roles of Neutralizers and Continuous Renal Replacement Therapy.

Hydrofluoric acid (HF), a dangerous inorganic acid, is widely used in various industries and in daily life. Chemical burns caused by HF exposure occur more frequently in some regions worldwide. It has been reported that some cases with HF burns can be lethal due to the hypertoxicity of HF. In this article, we present a case of a 24-year-old worker who suffered HF burns by 53% HF solution to his face, neck, and nasal cavity. This patient quickly developed electrolyte disturbance, that is, hypocalcemia, and hypopotassemia, and myocardial injury after exposure. Multiple measures had been taken to treat this patient, including fluid resuscitation, electrolyte replacement, timely wound treatment with neutralizers, and respiratory tract care. Moreover, continuous renal replacement therapy was also employed to remove fluoride in the circulatory system and rectify the electrolyte disturbance and acid-base imbalance. The patient smoothly pulled though and survived. High fluoride levels in the dialysate solution were confirmed, indicating that continuous renal replacement therapy is an effective and potentially lifesaving treatment for acute HF poisoning.

[Cloning of GmHSFA1 gene and its overexpression leading to enhancement of heat tolerance in transgenic soybean].

Heat shock transcription factors (HSFs) are important in regulating heat stress response by mediating expression of heat shock protein (HSP) genes in various plant species. In the present study, a novel GmHSFA1 with an ORF of 1,533 bp (full-length cDNA sequence of 1,781 bp) was cloned from soybean genome via comparative genomic approach and RACE (rapid amplification of cDNA ends). This gene encodes 510 amino acids consisting of a protein of 56.2 kDa (GenBank accession number: AY458843). Similar to other HSFs, GmHSFA1 has the basic modular structure including DBD, OD, NLS, and CTAD. BLAST analysis revealed the identity of 52.46% between amino acid sequences between GmHSFA1 and LpHSFA1 that has the highest similarity to GmHSFA1 in all HSFA1s in various plant species. The results from RT-PCR, Northern blotting, and transformation showed: 1) GmHsfA1 exhibited the constitutive expression patterns in different tissues of soybean; 2) The expression level of GmHsfA1 in transgenic plants was notably higher than that in non-transgenic plants; 3) Overexpression of GmHsfA1 activated transcription of GmHSP22 in transgenic plants under normal conditions and enhanced obviously expressions of GmHSP23 and GmHSP70 in transgenic plants under heat stress conditions; 4) Heat tolerant temperature (as high as 52 degrees C) of transgenic plants was remarkably higher than that of non-transgenic plants. These results preliminarily proved that the overexpression of GmHsfA1 possibly led to the notable enhancement of heat-tolerant level of transgenic plants by mediating the activation of transcription or improvement of expression of some GmHSPs in the GmHsfA1's downstream in transgenic plants, suggesting GmHSFA1 is a novel and functional heat shock transcription factor of soybean.

Hydrofluoric acid burns in the western Zhejiang Province of China: a 10-year epidemiological study.

BACKGROUND: Chemical burns caused by hydrofluoric acid (HF) frequently occur in the Western Zhejiang Province. This study aimed to investigate the epidemiological characteristics of HF burns within this region. METHODS: A 10-year retrospective analysis was conducted using data from all inpatients with HF burns. These patients were treated at the Department of Burns and Plastic Surgery at our hospital between January 2004 and December 2013. Information obtained for each patient included sex, age, occupation, burn location, burn cause, and the hazard category of the chemical which caused the burn. Data regarding wound site and size, accompanying injuries, serum electrolyte levels, operations, length of hospital stay, and mortality were also assessed. RESULTS: A total of 201 patients (189 males, 12 females; average age: 38.33 ± 10.57 years) were admitted due to HF burns. Over the 10-year period, the morbidity of HF burns in the past 10 years showed a gradual increase, which paralleled the development of local fluoride industries. Most HF injuries were work related and distributed in working-age patients. Aqueous HF solutions, especially highly concentrated ones, were the most common chemical cause of HF burns. Moreover, inappropriate operation, machine problems, and inadequate protection were identified as the leading causes of HF burns in the workplace. The burn area was <5% of TBSA in more than 90% of patients, and the most common burn sites were the head, neck, and upper extremities. Approximately 17% of patients underwent surgical operation. Accompanying injuries should be detected and treated correctly in a timely manner. Lastly, electrolyte imbalances, such as hypocalcaemia, hypomagnesaemia, and hypokalaemia, occurred frequently in patients with HF exposure; however, hyperkalaemia was not encountered in this study. CONCLUSION: Based on the epidemiological results for HF burns in this region, the related enterprises and local authorities should be encouraged to upgrade management policies and to provide necessary occupational hazard education and safety training for high-risk occupations within high-risk working populations. Furthermore, the enhancement of hazardous chemicals management is also needed. Finally, for patients with HF exposure, early and correct pre-hospital triage, treatment and consequent in-hospital treatment and procedures should also be improved.

Ten-year epidemiology of chemical burns in western Zhejiang Province, China.

BACKGROUND: Chemical burns occur frequently in western Zhejiang Province. This study documents the epidemiology of chemical burns in the region using burn data from a local specialized hospital. Results from this analysis will assist in the planning of prevention strategies for high-risk occupations and groups. METHODS: A 10-year retrospective analysis was conducted for all patients with chemical burns admitted to the Department of Burn and Plastic Surgery from January 2004 to December 2013. Information obtained for each patient included demographics (gender, age, occupation and education), location of the burn, cause of the burn, and categories of chemicals. Data regarding the season of admittance, prehospital treatment, wound site/size (area, region, and depth), accompanying injuries, operations, length of hospital stay and mortality were also assessed. RESULTS: A total of 690 patients (619 males, 71 females; average age: 30.6±12.4 years) were admitted to the department for chemical burns. Over the 10-year period, the incidence of chemical burns showed an increasing tendency. Chemical burns occurred most frequently in patients aged 20-59 years (94.79%). Most of the chemical burns were work-related, primarily in private enterprises (47.97%) and state-owned enterprises (24.93%). Operations (68.99%) and machine problems (17.26%) were the main causes of chemical burns in the workplace. With regard to burns caused by chemicals, most were caused by acids (72.01%), with hydrofluoric acid and sulphuric acid causing 51.45%. Most chemical burns occurred in the summer and autumn seasons (61.02%). The burn size was <10% of the total body surface area (TBSA) for 445 patients (64.49%), while only 26 patients (3.76%) had burns covering >40% TBSA. The most common burn sites were the upper extremities (31.57%), lower extremities (19.86%), and head and neck (28.83%). Most patients (581 (84.20%)) received water washing treatment on site immediately after exposure. The most common accompanying injuries included inhalation injury, ocular burns and digestive tract injury. The average hospital stay was 17.0±23.1 days (range 1-333 days). Surgery was performed in 146 patients (21.16%), and the overall mortality rate was 0.58%. CONCLUSIONS: Chemical burns are preventable. The high morbidity of chemical burns in western Zhejiang Province is related to the industrial structure of the area. Governmental management in the fields of production, transportation, and utilization of chemicals could be improved. Workplaces have the responsibility to provide safe work environments and equipment, as well as occupational education and safety training for high-risk work groups.

Early and Thorough Wound Debridement Is Crucial for Treatment of Patients With Thermal Burns and para-Chloronitrobenzene Poisoning.

para-Chloronitrobenzene (p-CNB) is one of the important chemicals with high liposolubility and oxidizing properties. Heated p-CNB liquid can cause thermal injury by absorption over skin and wound and even methemoglobinemia by conversion of hemoglobin into methemoglobin. Severe methemoglobinemia is a life-threatening condition that demands immediate treatment. It is very rare for individuals to be injured by heated p-CNB, but they should be carefully attended to because of the peculiarities of this kind of injury. In the past 10 years, we received 5 patients who were injured by heated p-CNB. In addition to the intravenous administration of methylene blue, prompt and thorough wound management played a crucial role in the treatment of these critically ill patients, indicating the potential value of sufficient information for the clinical practitioners. The purpose of this article is to report our experience in the management of patients with thermal burns and p-CNB poisoning.

Cloning, sequencing and salt induced expression of PEAMT and BADH in oilseed rape (Brassica napus).

Agriculture productivity is severely hampered by soil salinity, drought and other environmental stresses. Studies on stress-resistant plants (halophytes, xerophytes, accumulating plants for specific toxic ions) have illuminated some mechanisms of stress tolerance in plants at metabolic or molecular levels, which gave some clues on how to genetically engineer stress-tolerant crops. With the isolation of more stress-responsive genes, genetic engineering with modified expression of stress responsive genes may be an effective way to produce stress-tolerant crops. In the present report, two genes (PEAMT and BADH) encoding the corresponding key enzymes for choline and glycine betaine (an important osmoprotectant) biosynthesis in plants were isolated in oilseed rape, an important oil crop in the world. Effects of salt stress on their expression were studied with quantitative PCR and their potential use in the genetic engineering of oilseed rape was discussed.

Cloning and characterization of a non-TIR-NBS-LRR type disease resistance gene analogue from peach.

Cloning of plant disease resistant genes is greatly helpful for disease resistant breeding in plants and the insight of resistance mechanism. However, there are less relevant researches in peach [prunus persica (L.) Batch]. In this study, four NBS-LRR type resistance gene analogs (RGAs) were cloned from genomic DNA of peach. The PNBS2 fragment was also amplified from peach cDNA and the full-length cDNA of PNBS2 (PRPM1, GenBank accession no. AY599223) has been cloned. Sequence analysis indicated that the cDNA of PRPM1 is 3007 bp in length and that the contained ORF encodes for a polypeptide of 917 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has typical structure of non-TIR-NBS-LRR genes, with NB-ARC, LZ, LRR and transmembrane domains. Southern analysis indicated that the PRPM1 gene might be a single copy in peach genome. Northern blot and RT-PCR analysis showed that the expression of PRPM1 was not induced by salicylic acid (SA) in peach young leaves. The isolation of putative resistance genes from peach provided useful bases for studying the structure and function of peach disease-resistance relating genes and disease resistant genetic breeding in peach.

Management of a patient with thermal burns and para-chloronitrobenzene poisoning.

Para-chloronitrobenzene (p-CNB), a hazardous and toxic substance, is widely used as an intermediary in chemical industries. p-CNB can cause methaemoglobinaemia due to electron-withdrawing properties of the nitro and chlorine groups. We present a case of a 23-year-old man suffering from thermal burns and p-CNB poisoning. In this case, severe methaemoglobinaemia was caused by the absorption of p-CNB through the burn wounds. Despite active treatment, such as the antidote of methylene blue, the patient's methaemoglobinaemia progressed, with slowly increasing methaemoglobin (MetHb) level. This case highlights the complexity and difficulty of managing this type of injury. To our knowledge, this case can be the first case report describing methaemoglobinaemia induced by p-CNB in a patient with thermal burns.

A review of treatment strategies for hydrofluoric acid burns: current status and future prospects.

Hydrofluoric acid (HF), a dangerous inorganic acid, can cause severe corrosive effects and systemic toxicity. HF enters the human body via where it contacts, such as skin and mucosa, alimentary and respiratory tracts, and ocular surfaces. In the recent years, the incidence of HF burn has tended to increase over time. The injury mechanism of HF is associated primarily with the massive absorption of HF and the release of hydrogen ions. Correct diagnosis and timely treatment are especially important for HF burns. The critical procedure to treat HF burn is to prevent on-going HF absorption, and block the progressive destruction caused by fluoride ions. Due to the distinct characteristics of HF burns, the topical treatment, as well as systemic support, has been emphasised. Whereas, management of patients with HF burns remains a great challenge in some situations. To date, there has been no widely accepted protocol for the rescue of HF burns, partly due to the diversity of HF burns. This paper overviews the current status and problems of treatment strategies for HF burns, for the purpose of promoting the future researches and improvement.

pYEMF, a pUC18-derived XcmI T-vector for efficient cloning of PCR products.

A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate applications in the fields of molecular biology and genetic engineering.