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Bovine meat and milk factors (BMMF) having structural similarity to plasmids of Acinetobacter baumanni and some single stranded (ss) DNA viruses are free circulating replication-competent circular ssDNA molecules which were first isolated from serum of Bos taurus animals. Isolation of BMMF from tissues of multiple sclerosis as well as some cancers led researchers to conclude that the consumption of these agents through milk and meat have a positive correlation with these diseases and that the absence of such molecules in B. indicus animals could account to the low occurrence of cancer in those areas consuming products of indigenous animals. Hence, the present study was designed to investigate its presence in Deoni, one of the indigenous cattle breeds of India. The DNA isolated from milk samples were subjected to rolling circle amplification (RCA) using exo-resistant random primers. The products of RCA were then used as template for PCR for amplification of circular DNA. The amplicons obtained of size around 2000bp were cloned in E. coli and sequenced. The BLASTn, multiple sequence alignment and phylogenetic analysis of the obtained sequences were carried out to identify the similarities of these isolates. The sequence showed maximum homology with either BMMF2 or plasmids of Acinetobactor spp. Replication initiation protein (Rep) which showed similarity to bacterial plasmids was present in the sequence. This is the pioneer report on the presence of BMMF in milk of indigenous cattle of India. [ABSTRACT FROM AUTHOR]

Several years of research into the small circular DNA molecules called SPHINX and BMMF (SPHINX/BMMF) have provided information on several areas of research, medicine, microbiology and nutritional science. But there are still open questions that have not yet been addressed. Due to the unclear classification, evolution and sources of SPHINX/BMMF, a risk assessment is currently not possible. However, risk assessment is necessary as SPHINX/BMMF are suspected to be involved in the development of cancer and neurodegenerative diseases. In order to obtain an overview of the current state of research and to identify research gaps, a review of all the publications on this topic to date was carried out. The focus was primarily on the SPHINX/BMMF group 1 and 2 members, which is the topic of most of the research. It was discovered that the SPHINX/BMMF molecules could be integral components of mammalian cells, and are also inherited. However, their involvement in neurodegenerative and carcinogenic diseases is still unclear. Furthermore, they are probably ubiquitous in food and they resemble bacterial plasmids in parts of their DNA and protein (Rep) sequence. In addition, a connection with bacterial viruses is also suspected. Ultimately, it is still unclear whether SPHINX/BMMF have an infectious capacity and what their host or target is. [ABSTRACT FROM AUTHOR]

Chronic inflammation, linked to the presence of bovine milk and meat factors (BMMFs) and specific subsets of macrophages, results in oxygen radical synthesis and induction of mutations in DNA of actively replicating cells and replicating single stranded DNA. Cancers arising from this process have been characterized as indirect carcinogenesis by infectious agents (without persistence of genes of the agent in premalignant or cancers cells). Here, we investigate structural properties of pleomorphic vesicles, regularly identified by staining peritumor tissues of colorectal, lung and pancreatic cancer for expression of BMMF Rep. The latter represents a subgroup of BMMF1 proteins involved in replication of small single‐stranded circular plasmids of BMMF, but most likely also contributing to pleomorphic vesicular structures found in the periphery of colorectal, lung and pancreatic cancers. Structurally dense regions are demonstrated in preselected areas of colorectal cancer, after staining with monoclonal antibodies against BMMF1 Rep. Similar structures were observed in human embryonic cells (HEK293TT) overexpressing Rep. These data suggest that Rep or Rep isoforms contribute to the structural formation of vesicles. [ABSTRACT FROM AUTHOR]

Sixteen novel circular rep-encoding DNA sequences with high sequence homologies to previously described SPHINX and BMMF sequences were isolated for the first time from non-bovine foods (pork, wild boar, chicken meat, Alaska pollock, pangasius, black tiger shrimp, apple, carrot, and sprouts from alfalfa, radish, and broccoli). The phylogenetic analysis of the full-length circular genomes grouped these together with previously described representatives of SPHINX/BMMF group 1 and 2 sequences (eight in each group). The characterization of genome lengths, genes present, and conserved structures confirmed their relationship to the known SPHINX/BMMF sequences. Further analysis of iteron-like tandem repeats of SPHINX/BMMF group 1-related genomes revealed a correlation with both full-length sequence tree branches as well as Rep protein sequence tree branches and was able to differentiate subtypes of SPHINX/BMMF group 1 members. For the SPHINX/BMMF group 2 members, a distinct grouping of sequences into two clades (A and B) with subgroups could be detected. A deeper investigation of potential functional regions upstream of the rep gene of the new SPHINX/BMMF group 2 sequences revealed homologies to the dso and sso regions of known plasmid groups that replicate via the rolling circle mechanism. Phylogenetic analyses were accomplished by a Rep protein sequence analysis of different ssDNA viruses, pCRESS, and plasmids with the known replication mechanism, as this yielded deeper insights into the relationship of SPHINX/BMMF group 1 and 2 Rep proteins. A clear relation of these proteins to the Rep proteins of plasmids could be confirmed. Interestingly, for SPHINX/BMMF group 2 members, the relationship to rolling circle replication plasmids could also be verified. Furthermore, a relationship of SPHINX/BMMF group 1 Rep proteins to theta-replicating plasmid Reps is discussed. [ABSTRACT FROM AUTHOR]

Simple Summary: Since 2014, a special class of meat and milk-derived circular DNA agents—termed bovine meat and milk factors (BMMF)—has been linked to the process of indirect carcinogenesis in humans. These BMMF include members of the virus family Genomoviridae, as well as unclassified DNA elements exhibiting both viral and plasmid attributes. Initial detection of BMMF in European cattle samples led to the assumption that BMMF are exclusively present in aurochs-derived cattle and consequently in food produced thereof. In the meantime, a more widespread occurrence including water buffalo, sheep and goat milk has been shown. The aim of the present study was to broaden our knowledge concerning the BMMF occurrence in different and less domesticated ruminants like yaks, zebus and watusi cattle that serve for food production in various countries. The predominant detection of BMMF in fecal matter compared to blood provides novel clues regarding the origin and transmission routes of these entities finally reaching the food chain. Circular replicase-encoding single-stranded (CRESS) DNA viruses and other circular DNA agents are increasingly found in various samples and animals. A specific class of these agents—termed bovine meat and milk factors (BMMF)—has been supposed to act as a factor in indirect carcinogenesis in humans. Initial observations attributed the BMMF to European cattle breeds and foodstuffs produced thereof. In the present study, blood and fecal samples from African and Asian cattle were examined. BMMF molecules and genomoviruses were detected in all bovids under study. The majority (79%) of the 29 circular elements could be assigned to BMMF groups 1 and 2, whereas CRESS viruses of the family Genomoviridae accounted for the smaller part (21%). Two genomoviruses belong to the genus Gemykibivirus and one to the genus Gemykrogvirus. The remaining three might be considered as novel species within the genus Gemycircularvirus. The majority of all isolated molecules originated from fecal samples, whereas only three derived from blood. The results from this study expand our knowledge on the diversity and presence of circular DNA in different ruminants that serve for food production in many countries over the world. [ABSTRACT FROM AUTHOR]

Consumption of Eurasian bovine meat and milk has been associated with cancer development, in particular with colorectal cancer (CRC). In addition, zoonotic infectious agents from bovine products were proposed to cause colon cancer (zur Hausen et al., 2009). Bovine meat and milk factors (BMMF) are small episomal DNA molecules frequently isolated from bovine sera and milk products, and recently, also from colon cancer (de Villiers et al., 2019). BMMF are bioactive in human cells and were proposed to induce chronic inflammation in precancerous tissue leading to increased radical formation: for example, reactive oxygen and reactive nitrogen species and elevated levels of DNA mutations in replicating cells, such as cancer progenitor cells (zur Hausen et al., 2018). Mouse monoclonal antibodies against the replication (Rep) protein of H1MSB.1 (BMMF1) were used to analyze BMMF presence in different cohorts of CRC peritumor and tumor tissues and cancer-free individuals by immunohistochemistry and Western blot. BMMF DNA was isolated by laser microdissection from immunohistochemistry-positive tissue regions. We found BMMF Rep protein present specifically in close vicinity of CD68+ macrophages in the interstitial lamina propria adjacent to CRC tissues, suggesting the presence of local chronic inflammation. BMMF1 (modified H1MSB.1) DNA was isolated from the same tissue regions. Rep and CD68+ detection increased significantly in peritumor cancer tissues when compared to tissues of cancer-free individuals. This strengthens previous postulations that BMMF function as indirect carcinogens by inducing chronic inflammation and DNA damage in replicating cells, which represent progress to progenitor cells for adenoma (polyps) formation and cancer. [ABSTRACT FROM AUTHOR]

Isolation and characterization of circular replicase-encoding single-stranded (ss) DNA from animal, plant and environmental samples are rapidly evolving in virology. We detected 21 circular DNA elements, including one genomoviral sequence, in individual milk samples from domesticated Asian water buffaloes (Bubalus arnee f. bubalis). Most of the obtained genomes are related to Sphinx 1.76 and Sphinx 2.36 sequences and share a high degree of similarity to recently published circular DNAs—named BMMF (bovine meat and milk factors)—that have been isolated from commercial milk, as well as from bovine serum. Characteristic features such as rep genes, tandem repeats and inverted repeats were detected. These BMMF have recently been found to be present in taurine-type dairy cattle breeds descending from the aurochs (Bos primigenius). Importantly, the occurrence of BMMF has been linked to the higher incidence of colorectal and breast cancer in North America and Western Europe compared with Asia. This is the first report of circular ssDNA detected in milk from the domesticated form of the wild Asian water buffalo (B. arnee) belonging to the subfamily Bovinae. This novelty should be taken into account in view of the above-mentioned cancer hypothesis. [ABSTRACT FROM AUTHOR]