The prognostic value of CD8+ CTLs, CD163+ TAMs, and PDL1 expression in the tumor microenvironment of primary central nervous system lymphoma.

To explore immune cell infiltration and PDL1 expression in the tumor microenvironment (TME) of primary central nervous system lymphoma (PCNSL), we performed immunohistochemical staining on paraffin-embedded tumor tissues from 34 patients diagnosed with PCNSL. CD8 and CD163 positive cells were manually counted, and PDL1 expression was quantified by the H-score scoring method in the tumor center and around the tumor. The Kaplan-Meier method was used to analyze the prognostic value of the TME. We found obvious infiltration of CD8+ CTLs and CD163+ TAMs in the TME of PCNSL patients. And PDL1 was expressed in the tumor center as well as around the tumor. Survival analysis showed that high CD8+ CTLs levels and high intratumoral PDL1 expression were significantly correlated with longer OS. High CD8+ CTLs and CD163+ TAMs levels were associated with longer PFS.

Role and application of chemokine CXCL13 in central nervous system lymphoma.

Chemokine ligand 13 (CXCL13) and its chemokine receptor 5 (CXCR5) both play significant roles in the tumor microenvironment (TME). CXCL13 in cerebrospinal fluid (CSF) has recently been found to have significant diagnostic and prognostic value in primary and secondary central nervous system (CNS) diffuse large B-cell lymphoma (DLBCL), and the CXCL13-CXCR5 axis has been shown to play an important chemotactic role in the TME of CNS-DLBCL. In this review, we first describe the clinical value of CXCL13 in CSF as a prognostic and diagnostic biomarker for CNS-DLBCL. In addition, this review also discusses the specific mechanisms associated with the CXCL13-CXCR5 axis in tumor immunity of primary diffuse large B cell lymphoma of the central nervous system (PCNS-DLBCL) by reviewing the specific mechanisms of this axis in the immune microenvironment of DLBCL and CNS inflammation, as well as the prospects for the use of CXCL13-CXCR5 axis in immunotherapy in PCNS-DLBCL.

[The Prognostic Value of Prognostic Nutritional Index Combined with D-dimer in Patients with Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To explore the effects of prognostic nutritional index (PNI) combined with D-dimer on the prognosis of patients with newly diagnosed diffuse large B-cell lymphoma (DLBCL). METHODS: The clinical data of 73 DLBCL patients at initial diagnosis were retrospectively evaluated, and the optimal cut-off point of PNI and D-dimer were determined by ROC curve. The overall survival (OS) rate and progression-free survival (PFS) rate in different subgroups were compared using Kaplan-Meier survival curves. Univariate and multivariate Cox regression analysis was performed to identify the factors associated with OS. RESULTS: Compared with the low PNI group (PNI<44.775), the high PNI group (PNI≥44.775) had better OS (P =0.022) and PFS (P =0.029), the 2-year OS rates of the two groups were 55.6% and 78.3% respectively (P =0.041). Compared with the high D-dimer group (D-dimer≥0.835), the low D-dimer group (D-dimer<0.835) had better OS (P <0.001) and PFS (P <0.001), the 2-year OS rates of the two groups were 51.4% and 86.8% respectively (P =0.001). Meanwhile, patients in the high PNI+ low D-dimer group had better OS (P =0.003) and PFS (P <0.001) than the other three groups, the 2-year OS rate was statistically different from the other three groups (P <0.05). The multivariate analysis revealed that NCCN-IPI (HR =2.083, 95%CI : 1.034-4.196, P =0.040), PNI (HR =0.267, 95%CI : 0.076-0.940, P =0.040) and PNI+D-dimer (HR =9.082, 95%CI : 1.329-62.079, P =0.024) were the independent risk factors affecting OS in patients with DLBCL. Subgroup analysis showed that PNI, D-dimer, and PNI combined with D-dimer could improve the prognostic stratification in low and low-intermediate risk DLBCL patients. CONCLUSION: High PNI, low D-dimer and combination of high PNI and low D-dimer at initial diagnosis suggest a better prognosis in DLBCL patients.

CD39 (ENTPD1) in tumors: a potential therapeutic target and prognostic biomarker.

As a regulator of the dynamic balance between immune-activated extracellular ATP and immunosuppressive adenosine, CD39 ectonucleotidase impairs the ability of immune cells to exert anticancer immunity and plays an important role in the immune escape of tumor cells within the tumor microenvironment. In addition, CD39 has been studied in cancer patients to evaluate the prognosis, the efficacy of immunotherapy (e.g., PD-1 blockade) and the prediction of recurrence. This article reviews the importance of CD39 in tumor immunology, summarizes the preclinical evidence on targeting CD39 to treat tumors and focuses on the potential of CD39 as a biomarker to evaluate the prognosis and the response to immune checkpoint inhibitors in tumors.

Efficient delivery of a large-size Cas9-EGFP vector in porcine fetal fibroblasts using a Lonza 4D-Nucleofector system.

BACKGROUND: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips. METHODS: We firstly delivered a 13 kb Cas9-EGFP and a 3.5 kb pMAX-GFP vector into PFFs via 7 programs recommended by the Lonza basic protocol. We then tested 6 customized dual-electroporation programs for delivering the 13 kb plasmid into PFFs. In addition, we screened potential alternative electroporation buffers to the Nucleofector™ P3 solution. Finally, three CRISPR/Cas9-sgRNAs targeting Rosa26, H11, and Cep112 loci were delivered into PFFs with different single and dual-electroporation programs. RESULTS: Notably lower transfection efficiencies were observed when delivering the 13 kb vector than delivering the 3.5 kb vector in PFFs via the single-electroporation programs. The customized dual-electroporation program FF-113 + CA-137 exhibited higher transfection efficiencies than any of the single-electroporation programs using vessels (98.1%) or strips (89.1%) with acceptable survival rates for the 13 kb vector. Entranster-E buffer generated similar transfection efficiencies and 24-hour survival rates to those from the P3 solution, thus can be used as an alternative electroporation buffer. In the genome-editing experiments, the FF-113 + CA-137 and CA-137 + CA-137 programs showed significantly superior (P < 0.01) efficiencies to ones from the single-electroporation programs in vessels and strips. Entranster-E buffer produced higher indel efficiencies than the P3 buffer. CONCLUSIONS: We markedly increased the delivery efficiencies for a large vector via customized dual-electroporation programs using Lonza 4D-Nucleofector™ system, and Entranster-E buffer can be used as an alternative electroporation buffer to Nucleofector™ P3 buffer.

Highly Efficient A-to-G Editing in PFFs via Multiple ABEs.

Cytosine base editors (CBEs) and adenine base editors (ABEs) are recently developed CRISPR-mediated genome-editing tools that do not introduce double-strand breaks. In this study, five ABEs, ABE7.10, ABEmax, NG-ABEmax, ABE8e and NG-ABE8e, were used to generate A-to-G (T-to-C) conversions in five genome loci in porcine fetal fibroblasts (PFFs). Variable yet appreciable editing efficiencies and variable activity windows were observed in these targeting regions via these five editors. The strategy of two sgRNAs in one vector exhibited superior editing efficiency to that of using two separate sgRNA expression vectors. ABE-mediated start-codon mutation in APOE silenced its expression of protein and, unexpectedly, eliminated the vast majority of its mRNA. No off-target DNA site was detected for these editors. Substantial off-target RNA events were present in the ABE-edited cells, but no KEGG pathway was found to be significantly enriched. Our study supports that ABEs are powerful tools for A-to-G (T-to-C) point-mutation modification in porcine cells.

Frequent Gene Mutations and Their Possible Roles in the Pathogenesis, Treatment and Prognosis of Primary Central Nervous System Lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare extranodal non-Hodgkin lymphoma with poor prognosis. In recent years, the emergence of genetic subtypes of systematic diffuse large B-cell lymphoma has highlighted the importance of molecular genetics, but large-scale research on the molecular genetics of PCNSL is lacking. Herein, we summarize the frequent gene mutations and discuss the possible pathogenesis of PCNSL. Myeloid differentiation primary response gene 88 (MYD88) and CD79B mutations, which cause abnormal activation of noncanonical nuclear factor-κB, are prominent genetic abnormalities in PCNSL. They are considered to play a major role in the pathogenesis of PCNSL. Other genes, such as caspase recruitment domain family member 11 (CARD11), tumor necrosis factor alpha induced protein 3 (TNFAIP3), transducin (ß)-like 1 X-linked receptor 1, cyclin dependent kinase inhibitor 2A, PR domain zinc finger protein 1, and proviral insertion in murine malignancies 1, are also frequently mutated. Notably, the pathogenesis of immune insufficiency-associated PCNSL is related to Epstein-Barr virus infection, and its progression may be affected by different signaling pathways. The different mutational patterns in different studies highlight the heterogeneity of PCNSL. However, existing research on the molecular genetics of PCNSL is still limited, and further research into PCNSL is required to clarify the genetic characteristics of PCNSL.

[Prognostic Value of A New Risk Stratification Model (R2-ISS) in Newly Diagnosed Multiple Myeloma].

OBJECTIVE: To investigate the significance of a new risk stratification model (R2-ISS) in evaluating the prognosis of newly diagnosed multiple myeloma (MM). METHODS: Clinical data of 116 newly diagnosed MM patients admitted to Lanzhou University Second Hospital from June 2012 to March 2021 were retrospectively analyzed. According to R2-ISS, these patients were divided into four groups: low risk, low-intermediate risk, intermediate-high risk, and high risk. The significance of R2-ISS on prognosis of the patients was analyzed. RESULTS: Survival analysis showed that R2-ISS was associated with progression-free survival (PFS) (P=0.042) and overall survival (OS) (P=0.014). Cox univariate analysis showed that lactate dehydrogenase, serum calcium, serum creatinine, ß2-microglobulin, ISS, R-ISS, R2-ISS, t(4;14), and autologous hematopoietic stem cell transplantation (ASCT) were the influencing factors of OS in newly diagnosed MM patients (all P<0.05). Cox multivariate analysis showed that R-ISS, R2-ISS, and ASCT were independent risk factors affecting OS (all P<0.05). In addition, survival analysis of patients with different R2-ISS showed that ASCT improved PFS and OS. CONCLUSION: R2-ISS has prognostic value for newly diagnosed MM patients, while ASCT can improve the prognosis of patients with different R2-ISS.

Efficient Simultaneous Introduction of Premature Stop Codons in Three Tumor Suppressor Genes in PFFs via a Cytosine Base Editor.

Base editing is an efficient and precise gene-editing technique, by which a single base can be changed without introducing double-strand breaks, and it is currently widely used in studies of various species. In this study, we used hA3A-BE3-Y130F to simultaneously introduce premature stop codons (TAG, TGA, and TAA) into three tumor suppressor genes, TP53, PTEN, and APC, in large white porcine fetal fibroblasts (PFFs). Among the isolated 290 single-cell colonies, 232 (80%) had premature stop codons in all the three genes. C−to−T conversion was found in 98.6%, 92.8%, and 87.2% of these cell colonies for TP53, PTEN, and APC, respectively. High frequencies of bystander C−to−T edits were observed within the editing window (positions 3−8), and there were nine (3.01%) clones with the designed simultaneous three-gene C−to−T conversion without bystander conversion. C−to−T conversion outside the editing window was found in 9.0%, 14.1%, and 26.2% of the 290 cell colonies for TP53, PTEN, and APC, respectively. Low-frequency C−to−G or C−to−A transversion occurred in APC. The mRNA levels of the three genes showed significant declines in triple-gene-mutant (Tri-Mut) cells as expected. No PTEN and a significantly lower (p < 0.05) APC protein expression were detected in Tri-Mut cells. Interestingly, the premature stop codon introduced into the TP53 gene did not eliminate the expression of its full-length protein in the Tri-Mut cells, suggesting that stop codon read-through occurred. Tri-Mut cells showed a significantly higher (p < 0.05) proliferation rate than WT cells. Furthermore, we identified 1418 differentially expressed genes (DEGs) between the Tri-Mut and WT groups, which were mainly involved in functions such as tumor progression, cell cycle, and DNA repair. This study indicates that hA3A-BE3-Y130F can be a powerful tool to create diverse knockout cell models without double-strand breaks (DSBs), with further possibilities to produce porcine models with various purposes.

BMPR-IB gene disruption causes severe limb deformities in pigs.

In an attempt to generate g.A746G substitution in the BMPR-IB gene, we unexpectedly obtained BMPR-IB homozygous knockout piglets ( BMPR-IB -/-) and heterogeneous knockout piglets with one copy of the A746G mutation ( BMPR-IB -/746G) via CRISPR/Cas9 editing. Polymerase chain reaction (PCR) and sequencing revealed complex genomic rearrangements in the target region. All BMPR-IB-disrupted piglets showed an inability to stand and walk normally. Both BMPR-IB -/- and BMPR-IB -/746G piglets exhibited severe skeletal dysplasia characterized by distorted and truncated forearms (ulna, radius) and disordered carpal, metacarpal, and phalangeal bones in the forelimbs. The piglets displayed more severe deformities in the hindlimbs by visual inspection, including fibular hemimelia, enlarged tarsal bone, and disordered toe joint bones. Limb deformities were more profound in BMPR-IB -/- piglets than in the BMPR-IB -/746G piglets. Proteomic analysis identified 139 differentially expressed proteins (DEPs) in the hindlimb fibula of BMPR -IB -/746G piglets compared to the wild-type (WT) controls. Most DEPs are involved in skeletal or embryonic development and/or the TGF-ß pathway and tumor progression. Gene Ontology (GO) and protein domain enrichment analysis suggested alterations in these processes. Of the top 50 DEPs, a large proportion, e.g., C1QA, MYO1H, SRSF1, P3H1, GJA1, TCOF1, RBM10, SPP2, MMP13, and PHAX, were significantly associated with skeletal development. Our study provides novel findings on the role of BMPR-IB in mammalian limb development.

Recognition efficiency of atypical cardiovascular readings on ECG devices through fogged goggles.

In their continuing battle against the COVID-19 pandemic, medical workers in hospitals worldwide need to wear safety glasses and goggles to protect their eyes from the possible transmission of the virus. However, they work for long hours and need to wear a mask and other personal protective equipment, which causes their protective eye wear to fog up. This fogging up of eye wear, in turn, has a substantial impact in the speed and accuracy of reading information on the interface of electrocardiogram (ECG) machines. To gain a better understanding of the extent of the impact, this study experimentally simulates the fogging of protective goggles when viewing the interface with three variables: the degree of fogging of the goggles, brightness of the screen, and color of the font of the cardiovascular readings. This experimental study on the target recognition of digital font is carried out by simulating the interface of an ECG machine and readability of the ECG machine with fogged eye wear. The experimental results indicate that the fogging of the lenses has a significant impact on the recognition speed and the degree of fogging has a significant correlation with the font color and brightness of the screen. With a reduction in screen brightness, its influence on recognition speed shows a v-shaped trend, and the response time is the shortest when the screen brightness is 150 cd/m2. When eyewear is fogged, yellow and green font colors allow a quicker response with a higher accuracy. On the whole, the subjects show a better performance with the use of green font, but there are inconsistencies. In terms of the interaction among the three variables, the same results are also found and the same conclusion can be made accordingly. This research study can act as a reference for the interface design of medical equipment in events where medical staff wear protective eyewear for a long period of time.

Integrated analysis of miRNA and mRNA expression profiles in p53-edited PFF cells.

p53 is the most frequently mutated gene in human cancers, with over half of all tumors harboring mutation at this locus. R248 and R249 (corresponding to porcine R241 and R242), are among the hotspot mutations frequently mutated in liver, lung, breast, and some other cancers. In this study, p53 gene was knocked out or point-edited (R241 and R242 were converted to 241W and 242S) in porcine fetal fibroblast (PFF) cells via CRISPR-Cas9 technique. High throughput sequencing of miRNA and mRNA uncovered a total of 225 differentially expressed miRNAs (DEMs) and 738 differentially expressed genes (DEGs) in the p53 knockout (p53-KO) cells, and a total of 211 DEMs and 722 DEGs in the point-modified (p53-241W242S) cells. Totally 28 annotated DEMs were found to overlap between p53-KO/p53-WT and p53-241W242S/p53-WT miRNAs datasets, of which miR-34 c, miR-218, miR-205, miR-105-1, miR-105-2, miR-206, miR-224 and miR-429 play important roles in p53 regulatory network. Among the top 10 DEGs in p53-KO and p53-241W242S cells, most genes were reported to be involved in tumors, cell proliferation or cell migration. p53-KO and p53-241W242S cells showed a significantly higher (P < 0.01) proliferation rate compared with p53-WT cells. In conclusion, genetic modifications of p53 gene significantly affect the expression levels of a large number of genes and miRNAs in the PFF cells. The p53-edited PFF cells could be used as non-tumor cell models for investigating the p53 signaling network, and as donor cells for somatic nuclear transfer, with the aim to develop porcine models with the corresponding p53 mutations.Abbreviations: CRISPR-Cas9: Clustered regularly interspaced short palindromic repeats-associated protein 9; PFF: porcine fetal fibroblasts; SCNT: somatic cell nuclear transfer; RNA sequencing: small RNA sequencing and mRNA sequencing; DEGs: differentially expressed mRNAs; DEMs: differentially expressed miRNAs.

Semen quality improvement in boars fed with supplemental wolfberry (Lycium barbarum).

Wolfberry is well known for its health benefits in Asian countries. This study consisted of two experiments. In Experiment 1, nine boars were provided 40 g dried wolfberry per 100 kg body weight per day in addition to regular feed for 160 days (divided into 40 days phases: I, II, III, and IV) under step-down air temperature conditions. Controls (n = 9) were fed regular feed only. Significant (p < .05 or p < .01) or slight improvements in sperm progressive motility, total abnormality rate, sperm concentration, and total sperm per ejaculate were observed in the wolfberry group during phases II and III. No differences were observed in semen volume. After combining the data from phases II ~ IV, significant improvements were detected in all aforementioned traits (p < .05 or p < .01), except semen volume. In Experiment 2, the wolfberry group (n = 5) was fed wolfberry for 90 days and exhibited significantly reduced head, tail, and total abnormality rates (p < .05 or p < .01) in both fresh semen and semen stored for 72 hr at 17°C compared to the control group (n = 5). SOD activity also significantly increased in this group of boars. Collectively, the findings of this study suggest that wolfberry has a positive effect on boar semen quality.