Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

Best practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus, Paramecium bursaria Chlorella virus 1 (PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in its Chlorella variabilisNC64A host, and found that virus survivorship was highest (69.5 ± 16.5%) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9–30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts

\u3ci\u3eParamecium bursaria\u3c/i\u3e Chlorella Virus 1 Encodes a Polyamine Acetyltransferase

Background: PBCV-1 gene a654l encodes a protein with sequence similarity to GCN5 histone acetyltransferases. Results: A crystal structure of A654L bound to coenzyme A reveals how A654L acetylates polyamines, not histone lysines. Conclusion: A654L functions as a polyamine acetyltransferase. Significance: As the first viral polyamine acetyltransferase, A654L has a possible role in host polyamine catabolism in viral replication. Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5A˚ crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication. Includes Supplemental Material

\u3ci\u3eParamecium bursaria\u3c/i\u3e Chlorella Virus 1 Encodes a Polyamine Acetyltransferase

Background: PBCV-1 gene a654l encodes a protein with sequence similarity to GCN5 histone acetyltransferases. Results: A crystal structure of A654L bound to coenzyme A reveals how A654L acetylates polyamines, not histone lysines. Conclusion: A654L functions as a polyamine acetyltransferase. Significance: As the first viral polyamine acetyltransferase, A654L has a possible role in host polyamine catabolism in viral replication. Paramecium bursaria chlorella virus 1 (PBCV-1), a large DNA virus that infects green algae, encodes a histone H3 lysine 27-specific methyltransferase that functions in global transcriptional silencing of the host. PBCV-1 has another gene a654l that encodes a protein with sequence similarity to the GCN5 family histone acetyltransferases. In this study, we report a 1.5A˚ crystal structure of PBCV-1 A654L in a complex with coenzyme A. The structure reveals a unique feature of A654L that precludes its acetylation of histone peptide substrates. We demonstrate that A654L, hence named viral polyamine acetyltransferase (vPAT), acetylates polyamines such as putrescine, spermidine, cadaverine, and homospermidine present in both PBCV-1 and its host through a reaction dependent upon a conserved glutamate 27. Our study suggests that as the first virally encoded polyamine acetyltransferase, vPAT plays a possible key role in the regulation of polyamine catabolism in the host during viral replication. Includes Supplemental Material

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Comparative genomics, transcriptomics, and physiology distinguish symbiotic from free-living \u3ci\u3eChlorella\u3c/i\u3e strains

Most animal–microbe symbiotic interactions must be advantageous to the host and provide nutritional benefits to the endosymbiont. When the host provides nutrients, it can gain the capacity to control the interaction, promote self-growth, and increase its fitness. Chlorella-like green algae engage in symbiotic relationships with certain protozoans, a partnership that significantly impacts the physiology of both organisms. Consequently, it is often challenging to grow axenic Chlorella cultures after isolation from the host because they are nutrient fastidious and often susceptible to virus infection. We hypothesize that the establishment of a symbiotic relationship resulted in natural selection for nutritional and metabolic traits that differentiate symbiotic algae from their free-living counterparts. Here, we compare metabolic capabilities of 5 symbiotic and 4 free-living Chlorella strains by determining growth levels on combinations of nitrogen and carbon sources. Data analysis by hierarchical clustering revealed clear separation of the symbiotic and free-living Chlorella into two distinct clades. Symbiotic algae did not grow on nitrate but did grow on two symbiont-specific amino acids (Asn and Ser) on which the free-living strains did not grow. The use of these amino acids was exclusively affected by the presence/absence of Ca2+ in the medium, and the differences were magnified if galactose was provided rather than sucrose or glucose. In addition, Chlorella variabilis NC64A genomic and differential expression analysis confirmed the presence of abundant amino acid transporter protein motifs, some of which were expressed constitutively both axenically and within the host. Significantly, all 5 symbiotic strains exhibited similar physiological phenotypes even though they were isolated as symbionts from different host organisms. Such similarities indicate a parallel coevolution of shared metabolic pathways across multiple independent symbiotic events. Collectively, our results suggest that physiological changes drive the Chlorella symbiotic phenotype and contribute to their natural fitness. Includes Supplementary materials

Nitric oxide synthase inhibition results in synergistic anti-tumour activity with melphalan and tumour necrosis factor alpha-based isolated limb perfusions

Nitric oxide (NO) is an important molecule in regulating tumour blood flow and stimulating tumour angiogenesis. Inhibition of NO synthase by L-NAME might induce an anti-tumour effect by limiting nutrients and oxygen to reach tumour tissue or affecting vascular growth. The anti-tumour effect of L-NAME after systemic administration was studied in a renal subcapsular CC531 adenocarcinoma model in rats. Moreover, regional administration of L-NAME, in combination with TNF and melphalan, was studied in an isolated limb perfusion (ILP) model using BN175 soft-tissue sarcomas. Systemic treatment with L-NAME inhibited growth of adenocarcinoma significantly but was accompanied by impaired renal function. In ILP, reduced tumour growth was observed when L-NAME was used alone. In combination with TNF or melphalan, L-NAME increased response rates significantly compared to perfusions without L-NAME (0–64% and 0–63% respectively). An additional anti-tumour effect was demonstrated when L-NAME was added to the synergistic combination of melphalan and TNF (responses increased from 70 to 100%). Inhibition of NO synthase reduces tumour growth both after systemic and regional (ILP) treatment. A synergistic anti-tumour effect of L-NAME is observed in combination with melphalan and/or TNF using ILP. These results indicate a possible role of L-NAME for the treatment of solid tumours in a systemic or regional setting. © 2000 Cancer Research Campaig

Identification of an L-Rhamnose Synthetic Pathway in Two Nucleocytoplasmic Large DNA Viruses

Nucleocytoplasmic large DNA viruses (NCLDVs) are characterized by large genomes that often encode proteins not commonly found in viruses. Two species in this group are Acanthocystis turfacea chlorella virus 1 (ATCV-1) (family Phycodnaviridae, genus Chlorovirus) and Acanthamoeba polyphaga mimivirus (family Mimiviridae), commonly known as mimivirus. ATCV-1 and other chlorovirus members encode enzymes involved in the synthesis and glycosylation of their structural proteins. In this study, we identified and characterized three enzymes responsible for the synthesis of the sugar L-rhamnose: two UDP-D-glucose 4,6-dehydratases (UGDs) encoded by ATCV-1 and mimivirus and a bifunctional UDP-4-keto-6-deoxy-D-glucose epimerase/reductase (UGER) from mimivirus. Phylogenetic analysis indicated that ATCV-1 probably acquired its UGD gene via a recent horizontal gene transfer (HGT) from a green algal host, while an earlier HGT event involving the complete pathway (UGD and UGER) probably occurred between a protozoan ancestor and mimivirus. While ATCV-1 lacks an epimerase/reductase gene, its Chlorella host may encode this enzyme. Both UGDs and UGER are expressed as late genes, which is consistent with their role in posttranslational modification of capsid proteins. The data in this study provide additional support for the hypothesis that chloroviruses, and maybe mimivirus, encode most, if not all, of the glycosylation machinery involved in the synthesis of specific glycan structures essential for virus replication and infection

SMRT Sequencing of Paramecium Bursaria Chlorella Virus-1 Reveals Diverse Methylation Stability in Adenines Targeted by Restriction Modification Systems

Chloroviruses (family Phycodnaviridae) infect eukaryotic, freshwater, unicellular green algae. A unique feature of these viruses is an abundance of DNA methyltransferases, with isolates dedicating up to 4.5% of their protein coding potential to these genes. This diversity highlights just one of the long-standing values of the chlorovirus model system; where group-wide epigenomic characterization might begin to elucidate the function(s) of DNA methylation in large dsDNA viruses. We characterized DNA modifications in the prototype chlorovirus, PBCV-1, using single-molecule real time (SMRT) sequencing (aka PacBio). Results were compared to total available sites predicted in silico based on DNA sequence alone. SMRT-software detected N6-methyl-adenine (m6A) at GATC and CATG recognition sites, motifs previously shown to be targeted by PBCV-1 DNA methyltransferases M.CviAI and M. CviAII, respectively. At the same time, PacBio analyses indicated that 10.9% of the PBCV-1 genome had large interpulse duration ratio (ipdRatio) values, the primary metric for DNA modification identification. These events represent 20.6x more sites than can be accounted for by all available adenines in GATC and CATG motifs, suggesting base or backbone modifications other than methylation might be present. To define methylation stability, we cross-compared methylation status of each GATC and CATG sequence in three biological replicates and found ∼81% of sites were stably methylated, while ∼2% consistently lack methylation. The remaining 17% of sites were stochastically methylated. When methylation status was analyzed for both strands of each target, we show that palindromes existed in completely non-methylated states, fully-methylated states, or hemi-methylated states, though GATC sites more often lack methylation than CATG sequences. Given that both sequences are targeted by not just methyltransferases, but by restriction endonucleases that are together encoded by PBCV-1 as virus-originating restriction modification (RM) systems, there is strong selective pressure to modify all target sites. The finding that most instances of non-methylation are associated with hemi-methylation is congruent with observations that hemi-methylated palindromes are resistant to cleavage by restriction endonucleases. However, sites where hemi-methylation is conserved might represent a unique regulatory function for PBCV-1. This study serves as a baseline for future investigation into the epigenomics of chloroviruses and their giant virus relatives

Nationwide Outcome of Gastrectomy with En-Bloc Partial Pancreatectomy for Gastric Cancer

Background Radical gastrectomy is the cornerstone of the treatment of gastric cancer. For tumors invading the pancreas, en-bloc partial pancreatectomy may be needed for a radical resection. The aim of this study was to evaluate the outcome of gastrectomies with partial pancreatectomy for gastric cancer. Methods Patients who underwent gastrectomy with or without partial pancreatectomy for gastric or gastro-oesophageal junction cancer between 2011 and 2015 were selected from the Dutch Upper GI Cancer Audit (DUCA). Outcomes were resection margin (pR0) and Clavien-Dindo grade >= III postoperative complications and survival. The association between partial pancreatectomy and postoperative complications was analyzed with multivariable logistic regression. Overall survival of patients with partial pancreatectomy was estimated using the Kaplan-Meier method. Results Of 1966 patients that underwent gastrectomy, 55 patients (2.8%) underwent en-bloc partial pancreatectomy. A pR0 resection was achieved in 45 of 55 patients (82% versus 85% in the group without additional resection, P = 0.82). Clavien-Dindo grade = III complications occurred in 21 of 55 patients (38% versus 17%, P <0.001). Median overall survival [95% confidence interval] was 15 [6.8-23.2] months. For patients with and without perioperative systemic therapy, median survival was 20 [12.3-27.7] and 10 [5.7-14.3] months, and for patients with pR0 and pR1 resection, it was 20 [11.8-28.3] and 5 [2.4-7.6] months, respectively. Conclusions Gastrectomy with partial pancreatectomy is not only associated with a pR0 resection rate of 82% but also with increased postoperative morbidity. It should only be performed if a pR0 resection is feasible

Torsion of the Gallbladder

A 77-year-old woman was seen with progressive abdominal pain. A CT scan was made and showed a large gallbladder extending into the right lower abdomen. Ultrasound was performed but demonstrated no gallstones. Laparoscopy showed a tordated, necrotic gallbladder that was attached to the liver only by the cystic artery and cystic duct. Cholecystectomy was performed. Torsion of the gallbladder is a rare but clinically important condition in which the diagnosis seldom is made preoperatively. In radiological and clinical signs of cholecystitis without gallstones, this condition should be considered