Chemo-, Regio- and Stereoselective Preparation of (Z)-2-Butene-1,4-Diol Monoesters via Pd-Catalyzed Decarboxylative Acyloxylation.

(Z)-alkenes are useful synthons but thermodynamically less stable than their (E)-isomers and typically more difficult to prepare. The synthesis of 1,4-hetero-bifunctionalized (Z)-alkenes is particularly challenging due to the inherent regio- and stereoselectivity issues. Herein we demonstrate a general, chemoselective and direct synthesis of (Z)-2-butene-1,4-diol monoesters. The protocol operates within a Pd-catalyzed decarboxylative acyloxylation regime involving vinyl ethylene carbonates (VECs) and various carboxylic acids as the reaction partners under mild and operationally attractive conditions. The newly developed process allows access to a structurally diverse pool of (Z)-2-butene-1,4-diol monoesters in good yields and with excellent regio- and stereoselectivity. Various synthetic transformations of the obtained (Z)-2-butene-1,4-diol monoesters demonstrate how these synthons are of great use to rapidly diversify the portfolio of these formal desymmetrized (Z)-alkenes.

Laryngeal paraganglioma: The analysis of misdiagnosed cases and literature review.

Laryngeal paraganglioma (LP) is an exceptionally rare neuroendocrine tumor, underscoring importance of accurate identification to preclude misdiagnoses. In this review, we presented two typical misdiagnosed LPs, and offered reviews of LP cases reported over the preceding decade and all documented misdiagnosed LP cases. Furthermore, we systematically investigated the underlying causes of misdiagnosis and elucidated key points for effective differentiation. A retrospective analysis of 28 LP cases revealed a predominant occurrence in middle-aged women, with an average history of 25.1 months. Through an analysis of all misdiagnosed cases (n = 37), supraglottic LPs were frequently misidentified as laryngeal carcinomas and vascular tumors, while subglottic LPs were often misdiagnosed as thyroid cancers. And the occurrence of misdiagnosis resulted in delayed and inappropriate treatments, contributing to the deterioration of LP patients (14 cases, 37.8%). In conclusion, this review endeavored to heighten awareness of LPs, with the ultimate goal of advancing diagnostic precision and enhancing patient outcomes.

Circular economy strategies in supply chains, enhancing resource efficiency and sustainable development goals.

Eco-industrial parks are the real-world implementation of green supply chain management. There is a growing need to include the circular economy concept into supply chain management as a means of striking a better economic, social, and environmental balance, as the importance of the external sustainability of the supply chain is challenging. Using 357 questionnaires filled out by enterprises in China's eco-industrial parks, we examine the connections and causal relationships between resource efficiency, environmental impact, green supply chain management, and circular economy. To learn how a green supply chain's circular economy affects resource efficiency and environmental performance in the China region, this study makes use of the instrumental variable approach (structure equation model (SEM)). The results of this study indicate that environmentally responsible supply chain management and circular economy have beneficial effects on environmental performance and resource efficiency. The management of the GSC has a negative and small impact on economic performance, although each of the components is a substantial contributor to better performance in the environment. Conclusions from this study will assist those responsible for making decisions within supply chains in developing a plan that is useful for increasing a company's performance along economic and environmental dimensions. This study not only broadens our understanding of the factors that influence green supply chain management but also offers theoretical direction for the implementation of successful green production practices by businesses located in eco-industrial parks.

p38 MAPK involvement in the thermal stress response occurs via HSP27 and caspase3 in the large yellow croaker (Larimichthys crocea).

The p38 mitogen-activated protein kinase (p38 MAPK) is a multifunctional molecule that is involved in cellular response to various stressful stimuli. In the present study, the full-length cDNA sequence of p38 MAPK (Lcp38 MAPK) was identified from the large yellow croaker Larimichthys crocea, which encoded a polypeptide of 361 amino acid residues. The predicted Lcp38 MAPK protein contained a highly conserved Thr-Gly-Tyr (TGY) motif, a glutamate and aspartate (ED) site, a substrate binding site (Ala-Thr-Arg-Trp < ATRW>), and a serine/threonine kinase catalytic (S_TKc) domain characteristic of the MAPK family. The constitutive expression of Lcp38 MAPK was detected in most of the tissues examined with the strongest expression in intestine. Subcellular localization in LCK cells (kidney cell line from a L. crocea) revealed that Lcp38 MAPK existed in both the cytoplasm and cell nucleus. The expression of Lcp38 MAPK after temperature stress was tested in LCK cells. The results indicated that Lcp38 MAPK transcripts were significantly upregulated under both cold (10 °C) and heat stress (35 °C) (P < 0.05). Furthermore, the phosphorylation levels of p38 MAPK as well the transcriptional levels of heat shock protein 27 (HSP27) and caspase3 in LCK cells were significantly induced under thermal exposure (P < 0.05). However, the cold- and heat induced HSP27 and caspase3 expression was significantly suppressed by SB203580, a specific inhibitor of p38-MAPK (P < 0.05). These findings indicated that Lcp38 MAPK might be involved in the cellular stress response via HSP27 and caspase3 in large yellow croaker.

Identification of Two Clusters in Renal Pelvis Urobiome of Unilateral Stone Formers Using 2bRAD-M.

Urolithiasis is a common urological disease with increasing incidence and a high recurrence rate, whose etiology is not fully understood. The application of sequencing and culturomics has revealed that urolithiasis is closely related to the urinary microbiome (urobiome), shedding new light on the pathogenesis of stone formation. In this study, we recruited 30 patients with unilateral stones and collected their renal pelvis urine from both sides. Then, we performed 2bRAD-M, a novel sequencing technique that provides precise microbial identification at the species level, to characterize the renal pelvis urobiome of unilateral stone formers in the both sides. We first found that the urobiome in the stone side could be divided into two clusters (Stone1 and Stone2) based on distance algorithms. Stone2 harbored higher microbial richness and diversity compared to Stone1. The genera Cupriavidus and Sphingomonas were overrepresented in Stone1, whereas Acinetobacter and Pseudomonas were overrepresented in Stone2. Meanwhile, differential species were identified between Stone1 and Stone2. We further constructed a random forest model to discriminate two clusters which achieved a powerful diagnostic potential. Moreover, the urobiome of the non-stone side (Control1/2) was compared with that of the stone side (Stone1/2). Stone1 and Control1 showed different microbial community distributions, while Stone2 was similar to Control2 based on diversity analysis. We also identified differentially abundant species among all groups. We assumed that there might be different mechanisms of how microbiota contribute to stone formation in two clusters. Our findings might assist in the selection of suitable medical treatments for urolithiasis.

Case report: Novel mutations in the SPG11 gene in a case of autosomal recessive hereditary spastic paraplegia with a thin corpus callosum.

A 24-year-old man presented with insidious onset progressive gait disturbance and was finally diagnosed with autosomal recessive hereditary spastic paraplegia. Two novel mutations, including a frameshift mutation (c.5687_5691del) and a non-sense mutation (c.751C>T), were identified in the SPG11 gene of the patient through whole genome sequencing. The frameshift mutation of c.5687_5691del leads to a change in amino acid synthesis beginning with amino acid No. 1896 arginine and terminating at the 8th amino acid after the change (p. Arg1896MetfsTer8). The non-sense mutation (c.751C>T) causes the conversion of codon 251st encoding the amino acid Gln into a stop codon (p. Gln251Ter), resulting in premature termination of peptide synthesis. Although confirmation of compound-heterozygosity could not be performed, our findings enriched the phenotypic spectrum of SPG11 mutations related to hereditary spastic paraplegia.

The role of microbiome: a novel insight into urolithiasis.

Urolithiasis, referred to as the formation of stones in the urinary tract, is a common disease with growing prevalence and high recurrence rate worldwide. Although researchers have endeavoured to explore the mechanism of urinary stone formation for novel effective therapeutic and preventative measures, the exact aetiology and pathogenesis remain unclear. Propelled by sequencing technologies and culturomics, great advances have been made in understanding the pivotal contribution of the human microbiome to urolithiasis. Indeed, there are diverse and abundant microbes interacting with the host in the urinary tract, overturning the dogma that urinary system, and urine are sterile. The urinary microbiome of stone formers was clearly distinct from healthy individuals. Besides, dysbiosis of the intestinal microbiome appears to be involved in stone formation through the gut-kidney axis. Thus, the human microbiome has potential significant implications for the aetiology of urolithiasis, providing a novel insight into diagnostic, therapeutic, and prognostic strategies. Herein, we review and summarize the landmark microbiome studies in urolithiasis and identify therapeutic implications, challenges, and future perspectives in this rapidly evolving field. To conclude, a new front has opened with the evidence for a microbial role in stone formation, offering potential applications in the prevention, and treatment of urolithiasis.

Engineering Isopropanol Dehydrogenase for Efficient Regeneration of Nicotinamide Cofactors.

Isopropanol dehydrogenase (IPADH) is one of the most attractive options for nicotinamide cofactor regeneration due to its low cost and simple downstream processing. However, poor thermostability and strict cofactor dependency hinder its practical application for bioconversions. In this study, we simultaneously improved the thermostability (433-fold) and catalytic activity (3.3-fold) of IPADH from Brucella suis via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H) by 1.23 × 106-fold. When these variants were employed in three typical bioredox reactions to drive the synthesis of important chiral pharmaceutical building blocks, they outperformed the commonly used cofactor regeneration systems (glucose dehydrogenase [GDH], formate dehydrogenase [FDH], and lactate dehydrogenase [LDH]) with respect to efficiency of cofactor regeneration. Overall, our study provides two promising IPADH variants with complementary cofactor specificities that have great potential for wide applications. IMPORTANCE Oxidoreductases represent one group of the most important biocatalysts for synthesis of various chiral synthons. However, their practical application was hindered by the expensive nicotinamide cofactors used. Isopropanol dehydrogenase (IPADH) is one of the most attractive biocatalysts for nicotinamide cofactor regeneration. However, poor thermostability and strict cofactor dependency hinder its practical application. In this work, the thermostability and catalytic activity of an IPADH were simultaneously improved via a flexible segment engineering strategy. Meanwhile, the cofactor preference of IPADH was successfully switched from NAD(H) to NADP(H). The resultant variants show great potential for regeneration of nicotinamide cofactors, and the engineering strategy might serve as a useful approach for future engineering of other oxidoreductases.

Advances in Mechanism Research on Polygonatum in Prevention and Treatment of Diabetes.

Diabetes mellitus is a fast-growing disease with a major influence on people's quality of life. Oral hypoglycemic drugs and insulin are currently the main effective drugs in the treatment of diabetes, but chronic consumption of these drugs has certain side effects. Polysaccharides, saponins, flavonoids, and phenolics are the primary secondary metabolites isolated from the rhizomes of Polygonatum sibiricum Redouté [Asparagaceae], Polygonatum kingianum Collett & Hemsl [Asparagaceae], or Polygonatum cyrtonema Hua [Asparagaceae], which have attracted much more attention owing to their unique therapeutic role in the treatment and prevention of diabetes. However, the research on the mechanism of these three Polygonatum spp. in diabetes has not been reviewed. This review provides a summary of the research progress of three Polygonatum spp. on diabetes and its complications, reveals the potential antidiabetic mechanism of three Polygonatum spp., and discusses the effect of different processed products of three Polygonatum spp. in treating diabetes, for the sake of a thorough understanding of its effects on the prevention and treatment of diabetes and diabetes complications.

Parallel anomaly detection algorithm for cybersecurity on the highspeed train control system.

With the rapid development of the high-speed train industry, the high-speed train control system has now been exposed to a complicated network environment full of dangers. This paper provides a speculative parallel data detection algorithm to rapidly detect the potential threats and ensure data transmission security in the railway network. At first, the structure of the high-speed train control data received by the railway control center was analyzed and divided tentatively into small chunks to eliminate the inside dependencies. Then the traditional threat detection algorithm based on deterministic finite automaton was reformed by the speculative parallel optimization so that the inline relationship's influences that affected the data detection order could be avoided. At last, the speculative parallel detection algorithm would inspect the divided data chunks on a distributed platform. With the help of both the speculative parallel technique and the distributed platform, the detection deficiency for train control data was improved significantly. The results showed that the proposed algorithm exhibited better performance and scalability when compared with the traditional, non-parallel detection method, and massive train control data could be inspected and processed promptly. Now it has been proved by practical use that the proposed algorithm was stable and reliable. Our local train control center was able to quickly detect the anomaly and make a fast response during the train control data transmission by adopting the proposed algorithm.

Current advances in environmental stimuli regulating the glycyrrhizic acid biosynthesis pathway.

Glycyrrhizic acid, the main active ingredient of licorice, has good antibacterial, anti-tumor, anti-viral, anti-inflammatory, and immunostimulatory activities. However, the content of glycyrrhizic acid fluctuates greatly in different licorice cultivars, and production depends on plant sources, which greatly limits its development and applications. Therefore, increasing glycyrrhizic acid content has become a research priority. In recent years, regulation of the glycyrrhizic acid biosynthesis pathway has been analyzed, the downstream synthesis pathway in licorice has been fully investigated, some key genes have been cloned, polymorphisms have been studied, and the content of glycyrrhizic acid was shown to be regulated by environmental stimuli. This work has provided a basis for studying the regulation mechanism of the glycyrrhizic acid synthesis pathway. This review summarizes and discusses relevant research to provide a current understanding of the glycyrrhizic acid synthesis pathway and its regulation in licorice.

A High-Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis.

Chiral cyanohydrins are useful intermediates in the pharmaceutical and agricultural industries. In nature, hydroxynitrile lyases (HNLs) are a kind of elegant tool for enantioselective hydrocyanation of carbonyl compounds. However, currently available methods for demonstrating hydrocyanation are still stalled at precise, but low-throughput, GC or HPLC analyses. Herein, we report a chromogenic high-throughput screening (HTS) method that is feasible for the cyanohydrin synthesis reaction. This method was highly anti-interference and sensitive, and could be used to directly profile the substrate scope of HNLs either in cell-free extract or fermentation clear broth. This HTS method was also validated by generating new variants of PcHNL5 that presented higher catalytic efficiency and stronger acidic tolerance in variant libraries.

Roles of miRNA and lncRNA in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is currently the most malignant subtype of breast cancer without effective targeted therapies, which makes its pathogenesis an important target for research. A growing number of studies have shown that non-coding RNA (ncRNA), including microRNA (miRNA) and long non-coding RNA (lncRNA), plays a significant role in tumorigenesis. This review summarizes the roles of miRNA and lncRNA in the progression, diagnosis, and neoadjuvant chemotherapy of TNBC. Aberrantly expressed miRNA and lncRNA are listed according to their roles. Further, it describes the multiple mechanisms that lncRNA shows for regulating gene expression in the nucleus and cytoplasm, and more importantly, describes lncRNA-regulated TNBC progression through complete combining with miRNA at the post-transcriptional level. Focusing on miRNA and lncRNA associated with TNBC can provide new insights for early diagnosis and treatment-they can be targeted in the future as a novel anticancer target of TNBC.

Quantitative proteomics reveals the regulatory networks of circular RNA BTBD7_hsa_circ_0000563 in human coronary artery.

BACKGROUND: BTBD7_hsa_circ_0000563, which is located on chromosome 14, contains conserved binding sites with miR-155/130a and RNA-binding proteins according to bioinformatic prediction. We investigated the association of BTBD7_hsa_circ_0000563 expression in coronary artery segments with atherosclerotic stenosis and identified the proteome-wide BTBD7_hsa_circ_0000563-regulated proteins in human coronary artery. METHODS: The atherosclerotic grade and extent in coronary artery segments were determined by hematoxylin and eosin staining. BTBD7_hsa_circ_0000563 expression in eight coronary artery segments from one patient was quantified by RT-qPCR assay. A proteomic approach was adopted to reveal significant differences in protein expression between among four groups differing in their BTBD7_hsa_circ_0000563 expression levels. RESULTS: The RT-qPCR assay revealed that coronary artery segments with severe atherosclerotic stenosis had significantly low BTBD7_hsa_circ_0000563 levels. The proteomic analysis identified 49 differentially expressed proteins among the segment groups with different BTBD7_hsa_circ_0000563 expression levels, of which 10 were downregulated and 39 were upregulated with increases in the BTBD7_hsa_circ_0000563 level. The 10 downregulated proteins were P61626 (LYSC_HUMAN), P02760 (AMBP_HUMAN), Q02985 (FHR3_HUMAN), P01701 (LV151_HUMAN), P06312(KV401_HUMAN), P01624 (KV315_HUMAN), P13671 (CO6_HUMAN), P01700(LV147_HUMAN), Q9Y287(ITM2B_HUMAN), and A0A075B6I0 (LV861_HUMAN). The top 10 upregulated proteins were Q92552 (RT27_HUMAN), Q9UJY1(HSPB8_HUMAN), Q9Y235(ABEC2_HUMAN), P19022 (CADH2_HUMAN), O43837(IDH3B_HUMAN), Q9H479(FN3K_HUMAN), Q9UM22(EPDR1_HUMAN), P48681(NEST_HUMAN), Q9NRP0(OSTC_HUMAN), and Q15628(TRADD_HUMAN). CONCLUSION: BTBD7_hsa_circ_0000563 is involved in the atherosclerotic changes in human coronary artery segments. Verification, mechanistic, and function studies are needed to confirm whether patients with coronary artery disease would benefit from such personalized medicine in the future.

Analysis on the polymorphisms of site RS4977574, and RS1333045 in region 9p21 and the susceptibility of coronary heart disease in Chinese population.

BACKGROUND: Rs4977574 (A > G) and Rs1333045 (C > T) are both single nucleotide polymorphisms (SNPs) related with coronary artery disease, locating on chromosome 9p21.3. The study aimed to identify the correlation between rs4977574 and rs1333045 polymorphism genotypes and coronary heart disease (CHD) in a Chinese population. METHODS: Blood samples were collected from 855 subjects. A case-control study was used in this experiment, and 598 cases in the CHD group and 257 subjects in the control group were enrolled. Genotyping was identified by the Agena MassARRAY system. Statistical analysis was conducted by SPSS (Ver 16.0) and plink (Ver. 1.07, Shaun Purcell). Haplotype analysis was performed using Haploview software. RESULTS: Association analysis by plink indicated a significant difference in the allele distribution for single nucleotide polymorphisms between cases and controls (rs4977574 P = 0.003, rs1333045 P = 0.035). Fisher's exact test by plink proved that allele G may be associated with a higher risk of CHD (P = 0.003, odds ratio (OR) = 1.371) and the T allele was likely to reduce the risk of coronary events (P = 0.035, OR = 0.798). The serum levels of apolipoprotein A (ApoA) were higher in subjects with the AG + AA genotype of rs4977574 compared to those with the GG genotype (P = 0.028). In the dominant model of rs1333045, the levels of ApoA were higher and LDL levels were lower in the TC + TT genotype than in the CC genotype. CONCLUSIONS: The present study examined the association between the 9p21 chromosome rs4977574 and rs1333045 polymorphism genotypes and CHD in a population of Chinese patients. The G allele of rs4977574 and the C allele of rs1333045 are the susceptibility sites of CHD.

Current advances and outlooks in immunotherapy for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an incurable cancer resistant to traditional treatments, although a limited number of early-stage patients can undergo radical resection. Immunotherapies for the treatment of haematological malignancies as well as solid tumours have been substantially improved over the past decades, and impressive results have been obtained in recent preclinical and clinical trials. However, PDAC is likely the exception because of its unique tumour microenvironment (TME). In this review, we summarize the characteristics of the PDAC TME and focus on the network of various tumour-infiltrating immune cells, outlining the current advances in PDAC immunotherapy and addressing the effect of the PDAC TME on immunotherapy. This review further explores the combinations of different therapies used to enhance antitumour efficacy or reverse immunodeficiencies and describes optimizable immunotherapeutic strategies for PDAC. The concordant combination of various treatments, such as targeting cancer cells and the stroma, reversing suppressive immune reactions and enhancing antitumour reactivity, may be the most promising approach for the treatment of PDAC. Traditional treatments, especially chemotherapy, may also be optimized for individual patients to remodel the immunosuppressive microenvironment for enhanced therapy.

Circular RNA profile in coronary artery disease.

Circular RNAs (circRNAs) are potential biomarkers and therapeutic targets of coronary artery disease due to their high stability, covalently closed structure. And implied roles in gene regulation. The aim of this study was to identify and characterize circRNAs from human coronary arteries. Epicardial coronary arteries were removed during the autopsy of an 81-year-old man who died from heart attack. The natural history and histological classification of atherosclerotic lesions in coronary artery segments were analyzed by hematoxylin and eosin staining, and their circRNA expression profiles were characterized by RNA sequencing. RNA sequencing identified 1259 annotated and 381 novel circRNAs. Combined with the results of histologic examination, intersection analysis identified 54 upregulated and 12 downregulated circRNAs, representing 4.0% of the total number. Coronary artery segments with or without severe atherosclerosis showed distinctly different circRNA profiles on the basis of hierarchical clustering. Our results suggest that these 66 circRNAs contribute to the pathology underlying coronary artery atherosclerosis and may serve as diagnostic or therapeutic targets in coronary artery disease.

DNA methylation of antisense noncoding RNA in the INK locus (ANRIL) is associated with coronary artery disease in a Chinese population.

To explore the association between methylation of antisense non-coding RNA in the INK4 locus (ANRIL) and coronary artery disease (CAD) development. Methylation levels of ANRIL in 100 subjects with CAD and 100 controls were quantitatively analyzed using Sequenom MassARRAY. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was used to identify novel pathways. Our analyses indicated that 7 to 8 CpG sites within the 2nd CpG island located upstream of ANRIL, also known as cyclin-dependent kinase inhibitor 2B - antisense 1 (CDKN2B-AS1), are hyper-methylated in CAD subjects compared to controls (p = 0.034). The 40th CpG site within the 2nd CpG island located upstream of CDKN2B-AS1 was methylated to a lesser extent in CAD subjects compared to controls (p = 0.045). Both Pearson and Spearman analyses indicated that methylation levels were significantly associated with total cholesterol (r = 0.204, p = 0.004), fasting high-density lipoprotein cholesterol (r = 0.165, p = 0.020), and fasting low-density lipoprotein cholesterol (r = 0.265, p = 0.000). KEGG pathway analysis revealed a significant enrichment of genes associated with the tumor necrosis factor (TNF) signaling pathway. Among them, CCAAT/enhancer binding protein (C/EBPß) was identified as a key transcription factor that promotes expression of CDKN2B-AS1 through promotor interaction. DNA methylation of the ANRIL promoter was significantly associated with CAD development in our study. Our analyses suggest that C/EBPß is a key transcription factor that promotes CDKN2B-AS1 expression by directly interacting with the gene promotor mediated by TNF signaling.

The roles of PDGFRα signaling in the postnatal development and functional maintenance of the SMC-ICC-PDGFRα+ cell (SIP) syncytium in the colon.

BACKGROUND: The SIP syncytium in the gut consists of smooth muscle cells, interstitial cells of Cajal, and PDGFRα+ cells. We studied the fate of SIP cells after blocking PDGFRα receptor to explore the roles of PDGFRα signaling in the postnatal development and functional maintenance of the SIP syncytium. METHODS: Crenolanib was administered to mice from P0, P10, or P50. The morphological changes in SIP cells were examined by immunofluorescence. Protein expression in SIP cells was detected by Western blotting. Moreover, colonic transit was analyzed by testing the colonic bead expulsion time. KEY RESULTS: A dose of 5 mg(kg•day)-1 crenolanib administered for 10 days beginning on P0 apparently hindered the development of PDGFRα+ cells in the colonic longitudinal muscularis and myenteric plexus without influencing their proliferative activity and apoptosis, but this result was not seen in the colonic circular muscularis. SMCs were also inhibited by crenolanib. A dose of 7.5 mg(kg•day)-1 crenolanib administered for 15 days beginning on P0 caused reductions in both PDGFRα+ cells and ICC in the longitudinal muscularis, myenteric plexus, and circular muscularis. However, when crenolanib was administered at a dose of 5 mg(kg•day)-1 beginning on P10 or P50, it only noticeably decreased the number of PDGFRα+ cells in the colonic longitudinal muscularis. Crenolanib also caused PDGFRα+ cells to transdifferentiate into SMC in adult mice. Colonic transit was delayed after administration of crenolanib. CONCLUSIONS & INFERENCES: Therefore, PDGFRα signaling is essential for the development and functional maintenance of the SIP cells, especially PDGFRα+ cells.