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Driven by the need to reduce risk of SARS‐CoV‐2 infection and optimise use of health system resources, while maximising patient outcomes, the COVID‐19 pandemic has prompted unprecedented changes in cancer care. Some new or modified health care practices adopted during the pandemic will be of long term value in improving the quality and resilience of cancer care in Australia and internationally. The Cancer Australia consensus statement is intended to guide and enhance the delivery of cancer care during the pandemic and in a post‐pandemic environment. This article summarises the full statement, which is available at https://www.canceraustralia.gov.au/covid‐19/covid‐19‐recovery‐implications‐cancer‐care. The statement is informed by a desktop literature review and input from cancer experts and consumers at a virtual roundtable, held in July 2020, on key elements of cancer care that changed during the pandemic. It describes targeted strategies (at system, service, practitioner and patient levels) to retain, enhance and embed high value changes in practice. Principal strategies include: implementing innovative models of care that are digitally enabled and underpinned by clear governance, policies and procedures to guide best practice cancer care;enabling health professionals to deliver evidence‐based best practice and coordinated, person‐centred cancer care; andempowering patients to improve health literacy and enhancing their ability to engage in informed, shared decision making. Widespread adoption of high value health care practices across all levels of the cancer control sector will be of considerable benefit to the delivery of optimal cancer care into the future.

We investigated whether the toxicity‐associated receptor‐binding domain of the non‐immunogenic Escherichia coliheat‐stable enterotoxin (STh) as a fusion with a carrier protein and the inclusion of an appropriate spacer are critical factors for eliciting antibody responses against the native toxin. The immunological properties of three toxic and one non‐toxic fusion proteins, consisting of STh N‐terminally joined to the C‐terminus of the major subunit ClpG of E. coliCS31A fimbriae, were compared. In contrast to the non‐toxic hybrid STh with glycine and leucine simultaneously substituted for the receptor‐interacting Pro13and Ala14amino acids, the toxic chimeras responded by producing high serum levels of anti‐STh antibodies in immunized animals. On the other hand, only the toxic ClpG‐STh construct with the natural peptide 47KSGPESM53of Pro‐STh as spacer stimulated STh‐neutralizing responses against both native toxin and enterotoxigenic live E. colicells. Altogether, these findings suggest a close relationship between conformational similarity to the native structure of STh and the ability to elicit specific antibody responses against STh.

We have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH(2) or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

ABSTRACTWe have successfully used the major subunit ClpG ofEscherichia coliCS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. colihuman heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. colicells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH2or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. colicell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface ofE. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. colifor culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA(+) and dsbA(-) cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA(+) and dsbA(-) cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell.

Secretion of aerobactin is thought to play an important part in the virulence of invasive Escherichia coli also capable of synthesizing enterochelin. Why, despite its markedly lower affinity for iron than that of enterochelin, aerobactin proves to be the predominant active siderophore for bacterial growth in transferrin was investigated. We studied the action of two iron chelators, 2,2'-dipyridyl and transferrin, in expression of the aerobactin and enterochelin genes. Specifically, we describe the sequential localization of the two siderophores in the cell compartments during bacterial growth under different iron limitation conditions. Our results demonstrated that, whatever the exogenous iron-chelating agent used, aerobactin was rapidly excreted, whereas enterochelin accumulated early in periplasm before its very belated release into the external medium. This work also showed that the advantage of aerobactin over enterochelin in competition with transferrin was not due to (i) lack of enterochelin activity, (ii) a cell-bound aerobactin-dependent mechanism, (iii) antagonism between the two siderophores, and probably (iv) genetic preferential induction of aerobactin. We propose that the superiority of aerobactin in competing with transferrin for iron(III) was a consequence of its more rapid excretion with respect to enterochelin. In contrast to transferrin, 2,2'-dipyridyl induced a greater efficiency of enterochelin, possibly by a more permanent function as iron-binding compounds in the bacterial envelope. In summary, unlike aerobactin, enterochelin appears to be a weakly secreted high-affinity iron ligand.

Hydrogenase from Desulfovibrio desulfuricans (ATCC No. 27774) grown in unenriched and in enriched 61Ni and 57Fe media has been purified to apparent homogeneity. Two fractions of enzymes with hydrogenase activity were separated and were termed hydrogenase I and hydrogenase II. they were shown to have similar molecular weights (77,600 for hydrogenase I and 75,500 for hydrogenase II), to be composed of two polypeptide chains, and to contain Ni and non-heme iron. Because of its higher specific activity (152 versus 97) hydrogenase II was selected for EPR and Mössbauer studies. As isolated, hydrogenase II exhibits an "isotropic" EPR signal at g = 2.02 and a rhombic EPR signal at g = 2.3, 2.2, and 2.0. Isotopic substitution of 61Ni proves that the rhombic signal is due to Ni. Combining the Mössbauer and EPR data, the isotropic g = 2.02 EPR signal was shown to originate from a 3Fe cluster which may have oxygenous or nitrogenous ligands. In addition, the Mössbauer data also revealed two [4Fe-4S]2+ clusters iun each molecule of hydrogenase II. The EPR and Mössbauer data of hydrogenase I were found to be identical to those of hydrogenase II, indicating that both enzymes have common metallic centers.

The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia coli strains isolated from diarrheic or septicemic calves was studied. By electron microscopy studies, the E. coli surface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm) "fibrillar" fimbriae. E. coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31A fimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells. Conversely, E. coli K-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but was not able to hemagglutinate or adhere on intestinal cells. A single fimbrial subunit of 29 kilodaltons was observed when purified fimbriae from the 105-megadalton plasmid-containing E. coli K-12 strain was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 M guanidium hydrochloride from an S300 Sephacryl column. Western immunoblot analysis and the N-terminal sequence and amino acid composition of CS31A indicate structural and immunological relatedness between CS31A and K88 protein subunits.

The effect of exposure to carbon monoxide on the activity of the (Fe) hydrogenase from Desulfovibrio vulgarishas been determined. Concentrations of carbon monoxide which completely inhibit hydrogenase activity and induce formation of the axial g=2.06 EPR signal up to 0.8 spin/molecule do not cause irreversible inhibition of the (Fe) hydrogenase.

The effect of exposure to carbon monoxide on the activity of the (Fe) hydrogenase from Desulfovibrio vulgarishas been determined. Concentrations of carbon monoxide which completely inhibit hydrogenase activity and induce formation of the axial g=2.06 EPR signal up to 0.8 spin/molecule do not cause irreversible inhibition of the (Fe) hydrogenase.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.

To assess the role of the aerobactin-related system in the virulence of bovine opportunistic Escherichia coli, and to determine the stage(s) of the overall infectious process at which it is acting, germfree lambs were mixedly infected orally with two derivative strains of this bacterium differing in their ability (Iut+) or inability (Iut-) to express a functional aerobactin-mediated iron transport system. The Iut- strain was compared with the Iut+ strain for colonization of the gut, translocation to the mesenteric lymph nodes (MLN), and spread to other organs and to the body fluids of diassociated lambs. The Iut- mutant was found in smaller numbers in the duodenum, suggesting that aerobactin conferred a significant selective advantage for colonization of this intestinal segment. Although the two challenge strains translocated to MLN, the population level in the MLN was always higher for the Iut+ strain. Moreover, experimental infections resulted in recovery of only the Iut+ strain in the organs other than the MLN and in the body fluids. These results indicate a role for aerobactin in promoting systemic spread of the bacteria from the intestine. Direct evidence was obtained that aerobactin secretion occurred in vivo at both intestinal and extraintestinal sites of infection. In contrast to enterobactin, aerobactin was detected in the duodenum, jejunum, ileum, cecum, liver, spleen, kidney, urine, cerebrospinal fluid, and bile. The highest concentration of aerobactin was found in the urine, even when the samples were devoid of infecting bacteria. All of these findings suggest that aerobactin is released in vivo in a diffusible form and that it may be an important step in the production of disease by intestinal opportunistic E. coli.

The clpG gene coding for the CS31A subunit was localized on a 0.9-kb SphI fragment from the recombinant plasmid pAG315. This was established by testing the ability of subclones to hybridize with a 17-meric oligonucleotide probe obtained from N-terminal analysis of the CS31A subunit. The nucleotide sequence of the region coding for CS31A was determined. From primer extension analysis, two initiation translation start sites were detected. Two possible promoterlike sequences were identified; the ribosome binding site and the translation terminator are proposed. Inverted repeat sequences leading to the formation of possible hairpin structures of the transcripts were found on the 5' untranslated region of clpG. The deduced amino acid composition was in close agreement with the chemical amino acid composition and sequence match with the first 25 N-terminal amino acids from the published N-terminal sequence of the purified CS31A subunit. The clpG gene codes for a mature protein of 257 amino acids with a molecular size of 26,777 Da. An obvious homology was observed when the amino acid sequence of CS31A was compared with those of K88 and F41. This homology includes five different conserved sequences of up to 19 identical amino acids, which is associated with conserved proline. An extensive change in the CS31A region homologous to that identified to contain the K88 receptor binding site might be responsible for the functional divergence between CS31A and K88.

Two electrophoretic forms of the large subunit of the soluble periplasmic [NiFe] hydrogenase from Desulfovibrio gigashave been detected by Western analysis. The faster moving form co‐migrates with the large subunit from purified, active enzyme. Amino acid sequence and composition of the C‐terminal tryptic peptide of the large subunit from purified hydrogenase revealed that it is 15 amino acids shorter than that predicted by the nucleotide sequence. Processing of the nascent large subunit occurs by C‐terminal cleavage between His536and Val537, residues which are highly conserved among [NiFe] hydrogenases. Mutagenesis of the analogous residues, His582and Val583, in the E. colihydrogenase‐1 (HYD1) large subunit resulted in significant decrease in processing and HYD1 activity.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.

CS31A is a multimeric surface protein surrounding certain enterotoxigenic and septicemic bovine Escherichia coli strains. The possibility of using CS31A as a carrier of foreign antigenic determinants was investigated. Introduction of heterologous viral epitopes into the CS31A major subunit, ClpG, was successfully performed in the V3 region of the molecule which encodes for an immunodominant linear epitope. E. coli K-12 strains producing the hybrid CS31A molecules or the purified chimeric proteins were used for mice immunization. By using the C epitope derived from the S protein of the porcine transmissible gastroenteritis virus, significant antiviral antibody titers were elicited and seroneutralization of the virus was demonstrated, confirming that the molecular environment in V3 is favorable for antigen presentation. These results indicate that synthesis of CS31A hybrid proteins by the wild-type strain 31A could become a powerful tool for the development of oral vaccines directed against mucosal pathogens.

Muscle satellite cells (MuSCs) are the quiescent muscle stem cells required for adult skeletal muscle repair. The impact of environmental stress such as pollution on MuSC behavior remains unexplored. We evaluated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure, a ubiquitous and highly toxic pollutant, on MuSCs by combining in vivomouse molecular genetic models with ex vivostudies. While all MuSCs express the transcription factor PAX7, we show that a subset also express PAX3 and exhibit resistance to environmental stress. Upon systemic TCDD treatment, PAX3-negative MuSCs display impaired survival, atypical activation, and sporadic differentiation through xenobiotic aryl hydrocarbon receptor signaling. We further show that PAX3-positive MuSCs become sensitized to environmental stress when PAX3 function is impaired and that PAX3-mediated induction of mTORC1 is required for protection. Our study, therefore, identifies a functional heterogeneity of MuSCs in response to environmental stress controlled by PAX3.

We investigated whether the toxicity-associated receptor-binding domain of the non-immunogenic Escherichia coli heat-stable enterotoxin (STh) as a fusion with a carrier protein and the inclusion of an appropriate spacer are critical factors for eliciting antibody responses against the native toxin. The immunological properties of three toxic and one non-toxic fusion proteins, consisting of STh N-terminally joined to the C-terminus of the major subunit ClpG of E. coli CS31A fimbriae, were compared. In contrast to the non-toxic hybrid STh with glycine and leucine simultaneously substituted for the receptor-interacting Pro13 and Ala14 amino acids, the toxic chimeras responded by producing high serum levels of anti-STh antibodies in immunized animals. On the other hand, only the toxic ClpG-STh construct with the natural peptide 47KSGPESM53 of Pro-STh as spacer stimulated STh-neutralizing responses against both native toxin and enterotoxigenic live E. coli cells. Altogether, these findings suggest a close relationship between conformational similarity to the native structure of STh and the ability to elicit specific antibody responses against STh.

Two mutant strains (denoted as DCMUr-I and DCMUr-II) of the blue-green alga Aphanocapsa 6714, capable of growing in the presence of 10−5 M DCMU (an inhibitor of electron transport on the acceptor side of photosystem II), were isolated. The DCMU sensitivity of growth rates and of two photosystem II activities, O2 emission and fluorescence, was investigated in the mutant cells and compared with that of the wild type. The DCMU sensitivity of isolated thylakoids was also studied. The sensitivity of the mutant cells to other DCMU-type inhibitors (o-phenanthroline and atrazine) was tested. The results suggest that strain DCMUr-I resistance could be due to the acquisition of a cellular permeability barrier to DCMU, expressed only after an adaptation phase in the presence of the inhibitor. DCMUr-II resistance seems to be due primarily to an alteration of the thylakoid membranes of the photosynthetic apparatus itself.

The putative chaperone-like protein ClpE, required for biogenesis of the Escherichia coli capsule-like antigen CS31A, was compared with ten known periplasmic chaperones from E. coli, Klebsiella pneumoniae, Bordetella pertussis, Haemophilus influenzae and Yersinia pestis. The amino acid sequence alignment was superimposed onto the three-dimensional structure of the PapD chaperone of uropathogenic E. coli, and amino acid residues involved in maintaining the structure integrity of the suggested binding site were found identical in most of the 11 chaperones. Construction of a phylogenetic tree to investigate the relationship within the chaperone family has revealed interesting degrees of relatedness between the different proteins.