Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour. The chloroplast genome was determined to be 158,250 bp in length. It contained large single-copy (LSC) and small single-copy (SSC) regions of 84,929 bp and 12,663 bp, respectively, which were separated by a pair of 30,329 bp inverted repeat (IR) regions. The genome is predicted to contain 121 genes, including 78 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. The overall GC content of the genome is 38.1%. A phylogenetic tree reconstructed by 12 chloroplast genomes reveals that C. fargesii is mostly related to Catalpa. ovata and Catalpa. speciosa. This study identified the unique characteristics of the C. fargesii cp genome, which will provide a theoretical basis for species identification and biological research.In this study, we reported and characterized the complete chloroplast genome sequence of  Catalpa fargesii Bur. f. duclouxii (Dode) Gilmour belongs to the Catalpa genus of the Bignoniaceae family and is native to China. C. fargesii is distributed within the Yunnan-Guizhou plateau. It is recognized for its straight stems and high-quality timber, which is of high density and has high bending strength and hardness. These characteristics make it a valuable material for furniture production . The voucher specimens of C. fargesii were deposited at the herbarium of Southwest Forestry University (Voucher number: SWFU-2020-DQ01), and DNA samples were stored at the Key Laboratory for Forest Resources Conservation and Utilization in the Southwest Mountains of China Ministry of Education, Southwest Forestry University, Kunming, China. The total genomic DNA was extracted by using the Magnetic beads plant genomic DNA preps Kit . The genome skimming sequencing was conducted on the Illumina HiSeq 2000 Sequencing platform. Genome annotation was assembled with the program Geneious R8 . Finally, the chloroplast DNA sequence with complete annotation information was submitted to GenBank with accession number MW043481.The fresh leaves of C. fargesii is 158,250 bp in length, was 105 bp smaller than that of Catalpa. ovata . It was also 11 bp larger than Catalpa. Speciosa . The chloroplast genome has the usual quadripartite structure, featuring a LSC region , a SSC region , and a pair of IR . The overall GC content is 38.10% . respectively. A total of 121 functional genes were contained in the cp genome, including 78 protein-coding genes, 35 tRNA genes, and 8 rRNA genes.The complete cp genome of C. fargesii with respect to the other Bignoniaceae with fully sequenced chloroplast genomes, the complete C. fargesii chloroplast was used to reconstruct the phylogenetic relationships. With the chloroplast of Sesamum indicum  and S. indicum  as an out-group, 12 chloroplast genome sequences of Bignoniaceae, including Crescentia cujete (KT182634), Spathodea campanulata (MN106255), Oroxylum indicum (MN933930), C. ovata (MT186670), C. speciosa (MT319818), Podranea ricasoliana (MG831877), Tecomaria capensis (MG831880), Incarvillea arguta (MG763885), and Sesamum indicum (KC569603), aligned by the MAFFT version 7 programme (Katoh and Standley C. ovata and C. speciosa is most closely related to C. fargessi (C. fargesii has enough genetic information to distinguish it from other plants (Wang et al. C. fargesii and provides valuable genetic resources for the future development of chloroplast derived molecular markers. Our results provide fundamental information for further evolutionary and phylogenetic researches of C. fargesii. The chloroplast genome will contribute to the research and conservation of C. fargesii.To determine the phylogenetic location of fargessi , the chl


Bacillus velezensis strain BZR 517 is a prospective plant growth-promoting rhizobacterium with known biocontrol properties, which may be used to improve soil quality. The genome sequencing was conducted as part of new biological agent development in order to determine the biocontrol potential of the strain, including the production of biologically active compounds. Bacillus velezensis strain BZR 517 is a prospective plant growth-promoting rhizobacterium with known biocontrol properties, which may be used to improve soil quality. The genome sequencing was conducted as part of new biological agent development in order to determine the biocontrol potential of the strain, including the production of biologically active compounds. Bacillus subtilis, whose genome was first sequenced in 1997 by Kunst, was later classified as a complex taxon containing many phylogenetically close branches    , and liv, Spain) .B. velezensis strain BZR 517 was previously isolated from the roots of winter wheat grown in the Krylovsky district, Krasnodar Region, Russia, and was described as B. subtilis BZR 517 based on its morphological features and 16S rRNA gene sequence analysis. It was placed in the Departmental Collection of Useful Microorganisms for Agricultural Purposes (accession number RCAM 01728) as a plant growth stimulator  sequencing was conducted as part of the research dedicated to its antimicrobial and fungicidal activities for the purpose of developing a relevant commercial biocontrol agent. Details of the biomaterial preparation were described earlier , an N50 value of 341,123 bp, and a G+C content of 46.37%. Read preprocessing was performed using FastQC v.0.11.8 (https://github.com/s-andrews/FastQC), Trimmomatic v.0.39 (http://www.usadellab.org/cms/?page=trimmomatic), and Cutadapt v.2.5 (https://cutadapt.readthedocs.io/en/stable), and the genome was assembled with the SPAdes v.3.9.1 toolkit (https://cab.spbu.ru/software/spades). All software was deployed in separate Docker containers. The WGS assembly was annotated remotely on the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) using the best-placed reference protein set and GeneMarkS-2+ annotation software rev. 4.10 . The B. velezensis BZR 517 sequences obtained have been deposited in NCBI databases under the following accession numbers: PRJNA588983 (BioProject), SRX8162232 (SRA), and WKKV00000000 (GenBank).The complete project processing materials, including data, automation scripts, tools, relevant software launch parameters, and arguments, are available at the GitHub repository (


Porphyromonas gingivalis may affect genes and microglia (primary immune cells in the brain) to promote AD development. Genes for apolipoprotein, clusterin, CD33, triggering receptor expressed on myeloid cells-2 (TREM-2), tyrosine kinase binding protein (TYR-OBP), and complement receptors can affect microglia. Most of these genes can also be affected by P. gingivalis via its mastering of immune suppression. Besides, P. gingivalis can affect microglia directly in several ways. Taken together, genetic predisposition, P. gingivalis infection and microglia could promote neurodegeneration typical of that reported for AD.In late-onset Alzheimer disease (AD) pathogenesis, genes, infections and immunity could be significant factors. We have reviewed if the keystone periodontal pathogen  Cerebral inflammation in the form of activated microglia is a third major histopathological feature, but without a role in the neuropathological diagnosis of AD.Amyloid-beta (Aβ) plaque and phosphorylated tau (p-Tau) binding to neurofibrillary tangles (NFTs) are important neuropathological and diagnostic markers of Alzheimer’s disease (AD). Both lesions in their diverse peptide sizes (Aβ and p-Tau) can act as toxins in and outside cells l models . A seconP. gingivalis periodontal infection and microglia can interact to promote AD. Potential mechanisms for microglia affliction by P. gingivalis are listed in The aim of the present review is to discuss how genetic factors, P. gingivalis is a suspect pathogen, for details, see references [P. gingivalis has several virulence factors to promote brain inflammation and associated damage.Important in the relationship between ‘chronic’ periodontitis and AD is infection where ferences –5. An inP. gingivalis has initiated therapy towards reducing p-Tau peptide-related toxicity [P. gingivalis virulence with inhibitors of gingipains  [Institutionalized AD subjects show all forms of dental diseases , to co-exist in their dentition, and good oral health practices are unlikely to be a priority in their daily lives . Recent toxicity . This is3823404) .P. gingivalis are reported to digest the normal tau protein into nine fragments [Gingipains of ragments , and somragments . This main vivo and in vitro, having detrimental effects on tau [P. gingivalis W83 strain has shown the potential to contribute to tau phosphorylation at Ser396 in the in vivo wild-type mouse model [in vitro neuronal cell line model reported by Haditsch et al. [P. gingivalis infection with persistent gingipain expression. Liu et al. [P. gingivalis--infected microglial cells towards the site of infection, activation of the phosphoinositide 3-kinase/Akt (PI3K/AKT) pathway. Our own in house data show that purified P. gingivalis lipopolysaccharide (LPS) application to a neuroblastoma cell line, in vitro cell model also activated the PI3K/AKT pathway in which glycogen synthase kinases-3 beta (GSK-3β) mRNA expression increased. The importance here is that GSK-3β is one of the enzymes that phosphorylates tau suggesting that P. gingivalis plays an important role in the NFT lesion formation and subsequent pathophysiology of AD.Gingipains have been found to be neurotoxic s on tau . The capu et al.  observedP. gingivalis infected neurons in an in-vitro culture system with persistent expression of active gingipains. Following infection with live P. gingivalis (ATCC 33277) 25% of the neurons were lost in a time-dependent manner. Full-length tau was reduced in surviving cells with an increase in phosphorylation over time. This finding was related to loss of neuronal synapses and was comparable to features of associated neurodegeneration together with the presence of gingipains in AD autopsy brains. Accordingly, P. gingivalis can invade and survive in neurons and generate intra-neuronal gingipains that are proteolytically active, leading to neurodegeneration associated with AD.As mentioned, Haditsch et al.  reportedin vitro study that secreted gingipains from P. gingivalis induced microglial cell migration. This was likely achieved through endosomal signaling by protease-activated receptor 2 (PAR 2).Nonata and Nakanishi  found inP. gingivalis-infected microglial cells towards the site of infection, and initiated expression of proinflammatory mediators by activating PAR 2. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase/ERK pathway contributed to both cell migration and invoked an inflammatory response in microglia. Furthermore, PI3K/AKT pathway mRNA expression increased together with pro-inflammatory mediators such as IL-6, TNF-α and inducible nitric oxide synthase. The mRNA expression of the anti-inflammatory mediators interleukin 10 (IL-10), arginase-1 and IL-4 was not affected. The authors proposed that microglial cell migration was likely to have been associated with actin polymerization and may be necessary for invoking inflammatory responses in microglia following activation of PAR 2. Further observations by Liu et al. [P. gingivalis into the brain. Liu et al. [P. gingivalis-induced microglia cell migration was suppressed in the presence of the activated PAR 2 pathway. This provided proof of principle indicating that Rgp and Kgp were largely responsible for inducing migration of microglia in the brain. In the study by Dominy et al. [P. gingivalis reduced the extent of the brain infection established in mice. In addition, the small-molecule inhibitors blocked Aβ1-42 production, diminished neuroinflammation, and rescued neurons in the hippocampus.Liu et al. , attemptMatrix metalloproteinases (MMPs) have an important role in neuroinflammatory disorders including AD ,13. IncrP. gingivalis can induce the synthesis of MMPs in tissues and cells of the host. For example, cytokine and MMP expression in fibroblasts from peri-implantitis lesions were reported to be induced by P. gingivalis [P. gingivalis promoted invasion by induction of proMMP-9 and its activation [P. gingivalis activated protease-activated receptor 4 (PAR4) signaling pathways, causing proMMP-9 over-expression and invasion of oral squamous carcinoma cells [P. gingivalis does spread to the AD brain as shown experimentally in mice and in humans [P. gingivalis could contribute to the pool of MMPs in the brain.It is accepted that ngivalis . In addingivalis . In oraltivation . It was ma cells . Since Pn humans  it is plP. gingivalis extracts caused the destruction of connective tissue, contributing to the process of periapical disease by activating pro-MMP-2 as well as by inactivating TIMP-1 and TIMP-2. In another study using human gingival fibroblasts, P. gingivalis LPS differentially modulated the expression of MMP-1, −2, and −3 and TIMP-1 [P. gingivalis suppresses TIMPs activity in the brain, to dysregulate the MMP pool in AD patients. Again further research is warranted to clarify this possibility.Tissue inhibitors of metalloproteinases (TIMPS) can modulate the activity of MMPs . This isd TIMP-1 . AlternaP. gingivalis in apolipoprotein E−/- (ApoE−/-) mice, the host releases copious amount of IL-10 in the serum. However, the bacterium itself still spreads to the brain and encounters microglia, which as a result become activated [−/- mice remains inadequate for microglia to return to the resting state [Microglia comprise 10% of the total brain cells. They are resident macrophages and the brain’s primary innate immune cells responding to diverse stimuli ,b). Theyctivated . It appeng state ,26. Receng state . This maIt seems the severity of AD and its progression may be linked to chronic inflammation ,29. It iin situ brain mapping, and detected nine transcriptionally distinct stages of >76,000 microglial cells in mice expressing unique sets of genes. Some of these genes were upregulated in microglia-surrounding Aβ plaques [Microglia are not uniform cells, and this is why only fragments of microglia are seen in tissue sections following their immuno/fluorescence/histochemistry reactivities ,b). Acti plaques . Microgl plaques .Microglia can also develop functional defects as seen in other neurodegenerative disorders . During −/- mice brains that were from a mono-P. gingivalis-infected group [P. gingivalis infection [P. gingivalis may exert on microglia, which are less appreciated.It is noteworthy that gingipains have been detected in microglia , and in ed group . Mice brnfection . HoweverP. gingivalis infection of the brain, the impressive immune subversion of this bacterium in cleaving receptors challenges this very function as discussed below.Genome-Wide-Association Studies (GWAS) have identified several susceptibility genes expressed by microglia in AD. Genome-wide meta-analysis of clinically diagnosed AD and AD-by-proxy  found associated genes to be strongly expressed in immune-related tissues (spleen and liver), and cell types such as microglia . Other Gclusterin gene was identified as an important risk locus for AD with the three SNPs  showing a statistically significant relationship with the disease [The  disease ,47. Clus disease . It is p disease . Due to  disease . CLU is  disease .CR1 gene are associated with increased likelihood of AD [CR1 gene defect in AD [P. gingivalis mediates immune subversion in relation to CR1 [P. gingivalis.It has been reported that SNPs rs3818361 and rs6656401 of the od of AD . This sun to CR1 . This maP. gingivalis also binds [P. gingivalis cell surface components such as LPS may provide additional beneﬁts to this prominent periodontal pathogen in bioﬁlm formation and in escaping complement-mediated killing [P. gingivalis binding to them and is also a general clearance receptor for pathogens [P. gingivalis is either able to cleave CD33 from the surface membrane of cells or down-regulate functional cell surface receptors on myeloid cells. If this was the case, then the CD33 receptor expression would be affected in a similar way on microglia.CD33 appears to have an important role in Aβ clearance and other neuroinflammatory pathways in the brain aided by microglia . CD33 beso binds . Sialyla killing . CR1 is athogens . HoweverTREM-2 gene codes for a protein in the brain that is expressed on microglial cells and is also involved in removing degenerated tissue, including remnants from neuroinflammation [ε4 allele [P. gingivalis significantly down-regulated TREM-2 expression in microglia [The ammation . The TRE4 allele ,60. TREM4 allele . Interesicroglia . Lack oficroglia .TYR-OBP has been identified as a key regulator among genes involved in phagocytosis [TREM-2, as determined from networks involved in immune and microglia-specific modules disrupted in AD brains [P. gingivalis activity is little understood. Further research is needed to clarify if P. gingivalis can affect TREM-2 signaling through TYR-OBP.ocytosis . It is aD brains . The assP. gingivalis has been proposed to exploit complement receptors 1 and 3 for evading innate immune clearance [P. gingivalis-induced complement activation in ApoE−/- mice brains has been investigated [P. gingivalis, oral infection) and control groups exhibited strong intracellular labeling with complement components/opsonins from C3 and C9, due to ongoing biosynthesis and activation. Further, Poole et al. [P. gingivalis was able to access the ApoE−/- brain and contribute to the development of AD inflammatory pathology through mechanisms involving acute-phase proteins, cytokines and the complement cascade where neurons would be attacked. It has since been shown that ApoE binds to activated C1q and that the resulting C1q-ApoE complex becomes a common player to affect brain inflammation [learance ,66. Actistigated . Microgle et al.  showed tammation . Thus, iammation . As mentammation .P. gingivalis. Several animal studies have shown that LPS administered directly into the peritoneum of the brain initiates neuroinflammation in the form of microglial cell activation [e.g. LPS is one of the major virulence factors of ion e.g. . Researcion e.g. ,70. In tion e.g.  the micrP. gingivalis LPS also causes a higher increase in IL-1β. Interestingly, the most prominent induction of IL-1β was detected in MHC II (+) microglia from aged mice [P. gingivalis LPS induced cognitive dysfunction, mediated by neuronal inflammation via activation of the TLR4 signaling pathway in C57BL/6 mice. Both microglia and astrocytes in the cortex and hippocampus were activated. Accordingly, age-associated priming of microglia seems to have a central role in exaggerated inflammation induced by activation of the peripheral immune system. IL-1β is also implicated in synaptic loss [P. gingivalis LPS has been reported in the human brain, thus suggesting it might activate brain microglia participating in brain inflammation [in vitro stimulation study with ultrapure P. gingivalis LPS in rats that resulted in classical and alternative activation of rat brain microglia and the concomitant release of cytokines and chemokines [In a study by Henry et al.  peripherged mice . In anotged mice  found thtic loss ,75, promtic loss  by stimutic loss . Last, bemokines .P. gingivalis-LPS has this capacity is not known. P. gingivalis causes an imbalance in M1/M2 activation in macrophages, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis [P. gingivalis or P. gingivalis-derived LPS-induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL-4. Interestingly, resveratrol has been found to reduce inflammatory damage and promote microglia polarization to the M2 phenotype in LPS-induced neuroinflammation [Microglia, being influenced by their environment, can assume a diversity of phenotypes and can change functions aimed to maintain homeostasis. Like their macrophage ‘cousins’, microglia show unique features with regard to phenotype polarization. As mentioned, they can be stimulated by LPS and IFN-ɣ to develop into an M1 phenotype for expression of proinflammatory cytokines, or by IL-4/IL-13 to an M2 phenotype for resolution of inflammation and tissue repair . Whetherdontitis . These aammation .P. gingivalis LPS. The mean amount of TNF-α and IL-1β after exposure to conditioned medium from P. gingivalis LPS-stimulated macrophages were significantly higher than after treatment with P. gingivalis LPS alone. This indicated that leptomeningeal cells could transduce inflammatory signals to microglia in the deeper brain areas, which in turn initiated neuroinflammation.The leptomeninges  plays a role as secretory cells, which transduce systemic inflammatory signals into the CNS . In studP. gingivalis in wild-type mice transferred P. gingivalis to the brain where the bacterium and its proteases (gingipains) were detected within intra-nuclear and peri-nuclear locations of microglia, astrocytes, neurons, and extracellular spaces [P. gingivalis infection with persistent gingipain expression with ongoing neurodegeneration.Repeated chronic oral administration of r spaces . Microglr spaces  have pror spaces  confirmer spaces  for p-TaP. gingivalis. The latter may affect microglia in both direct and indirect ways. Whether other putative periopathogens and even intestinal bacteria also affect microglia of the AD brain remain to be tested. Astrocytes, which are macroglia, can also be activated by P. gingivalis. Such activation may have toxic effects on neurons. The chronic nature of low-level infections such as ‘chronic’ periodontitis and associated byproducts, e.g. endo/exotoxins and cytokines could affect susceptible brains’ defense capacity to a point where microglia involved in brain protection become adversely affected. Whether microglia will ‘remember’ inflammation caused by P. gingivalis and develop ‘tolerance’ to it, requires further research. However, it is plausible to suggest that once microglia are primed by P. gingivalis exposure, there remains the possibility of developing tolerance through the mastery of innate immunity manipulation by this bacterium, which may be the result of inadequate clearance of cellular debris (Aβ) from the AD brain.GWAS have indicated that genes, pathogens and the immune system act together to generate AD. In addition, neuroinflammation plays a pivotal role and this has made scientists ask the question if AD is an infectious disease. In this complex interaction of different players, microglia seem to be important in the host defense against invasion of the keystone periodontopathogen


Apis. In regions such as South America, Australia, and Southeast Asia, information on stingless bee bee bread is mainly sought to promote the meliponiculture industry for socioeconomic development. This review aims to highlight the physicochemical properties and health benefits of bee bread from the stingless bee. In addition, it describes the current progress on identification of beneficial microbes associated with bee bread and its relation to the bee gut. This review provides the basis for promoting research on stingless bee bee bread, its nutrients, and microbes for application in the food and pharmaceutical industries.Stingless bee-collected pollen (bee bread) is a mixture of bee pollen, bee salivary enzymes, and regurgitated honey, fermented by indigenous microbes during storage in the cerumen pot. Current literature data for bee bread is overshadowed by bee pollen, particularly of honeybee  Bees have a unique social life. In a social bee community, the queen dominates the reproduction and has a morphology different from other bees. The colonies live for many years  . StingleBee products are crucial for bee survival, and it is acquired for medicine, offering cosmetic, and everyday uses since 8000 BC . GradualBee bread is one the bee by-products made from pollen collected by the bee added with nectar and bee salivary enzymes before it undergoes lactic acid fermentation in beehives . Bee breConsumer interest and demand for natural products have influenced in-depth research for bee bread nutritional properties. Bee bread is rich in carbohydrate, protein, and lipids and contain other micronutrients such as minerals, vitamins, phenolic compounds, and essential amino acids . It possApis spp.) has been continuously reported [Research on bee pollen from European honeybee , which are essential for plant reproduction. It is either distributed through the wind (“anemophilous”—wind-pollinated) or by an insect (“entomophilous”—insect-pollinated). The global production of pollens was estimated as 1.36 million kg per year with China, Australia, and Argentina as the biggest contributors .Heterotrigona itama prefers foraging plants closest to their hive especially from white- and cream-coloured flowers [The process of stingless bee making bee bread begins from plant pollen, which is illustrated in  flowers ,21,22 wi flowers .Mimosa pudica [Mimosa caesalpiniaefolia [During foraging, forager bees collect nectar and store it in their honey stomach while their bodies are covered in pollen dust. Pollen profile collected by different stingless bees species has been documented in Southeast Asia ,26,27,28a pudica ,26 and Miaefolia .Apis. Bee pollen is acquired using a pollen trap installed at the hive entrance, which strips the pollen from the bee′s leg by forcing the bees to crawl through a small tight hole [As the bees collect pollens, they use their salivary enzymes (amylase and glucosidase)  and honeght hole .If the bee pollen is not harvested, the worker bees pack the bee pollen inside a cerumen pot (for stingless bee)  or honeysambura” is 247 USD/kg [A single stingless bee colony could produce up to 6 kg of bee bread per year depending on the species. In Brazil, the maximum price for bee bread, locally known as “7 USD/kg . In Mala7 USD/kg .Because stingless bee′s bee bread is stored in cerumen pot, its acquisition is different from those of honeybee. Honeybee bee bread is acquired either through manual extraction  or usageFlower pollen undergoes different development stages to become the end product known as bee bread. The biochemical profiles of flower pollen, bee pollen, and bee bread  are diffAloe greatheadii var. davyana and Helianthus anthuus (sunflower) showed that bee bread has significantly higher water and carbohydrate content, but lower protein, lipid, and fatty acid content than its fresh pollen [Comparing the chemical composition of monofloral pollens is more enticing . For exah pollen ,45. Flowh pollen ,45. Howeh pollen ,45.Melipona asilvai, M. quadrifasciata anthidioides, M. scutellaris, M. subnitida, Tetragona clavipes, and Plebeia sp. bee pollen. The pH for stingless bee bee bread will be discussed in the later section.Perhaps the noticeable change between bee pollen and bee bread is the pH. Bee bread is more acidic compared to bee pollen. Studies on pH changes of stingless bee pollen and bee bread are still lacking. However, when looking upon European bee pollen pH, it shows to be significantly reduced from pH 4.7 to 3.97 after transformation into bee bread . Duarte Scaptotrigona postica. A recent study also showed bee bread digestibility equivalent to those of fresh pollen even after consumption and digestion by Apis mellifera scutellata [Fermentation not only produces chemical changes in bee bread but also has been speculated to improve bee bread digestibility and bioavailability by degrading the outer pollen layer ,49,50. Nutellata . In thisNesocodon mauritianum) has purple pollen. Pollen colour tends to change into black once stored in hive due to oxidation [A stingless bee can carry a pollen weight of 10.9 mg  and the xidation . Bee proxidation , which rPollen wall has three different layers. The outermost layer is the pollenkitt followed by the outer wall (exine) and inner wall (intine). The exine consists of sporopollenin, while the intine is composed of cellulose and pectin . The exiBee bread contains accumulated pollen grains from various flowering plants. Although analysis of pollen colour could give an insight into its floral origin, a single pollen colour does not indicate monofloral source . This isMelissopalynology is the microscopic study of pollen grains in bee products such as honey, pollen, propolis, and royal jelly . Pollen Pollen morphology is species-specific; therefore, it provides valuable information on the pollen’s botanical origin and plants preferentially visited by the bees ,63. ThisTetragonula angustula, Ptilotrigona and Frieseomelitta doederlini, and F. varia produce sweet bee bread, whereas Melipona and Scaptotrigona produce bitter bee bread [The taste of bee bread is different across species. For example, ee bread ,64. To ree bread .Apis mellifera pollen to be considered as an international standard. However, unlike bee pollen, bee bread standard has yet to be articulated. Due to this setback, researchers opt to compare the research data of stingless bee bee bread to the international standard of bee pollen Apis spp. [Importantly, 250 different compounds comprise bee bread, and these are macro- and micronutrients, vitamins, amino acids, fatty acids, and phenolic compounds . Bee brepis spp. .It is challenging to generalise bee bread nutritional values. Several factors including botanical and geographical origin, climatic condition, soil type, beekeepers’ activities, or storage treatments in commercial production ,70 contrBee bread has shown to possess high moisture content. It is attributed to the pollen’s hygroscopic properties, which attracts water from the environment. Bee bread accumulates water from the surrounding and also through the addition of bee saliva and honey resulting in a sticky end product. Storage in closed pots prevents water loss from bee bread . AccordiBecause pollen is hygroscopic, bee bread requires adequate drying parameters to achieve the desired moisture content. Different preservation methods are utilised in the beekeeping industry to preserve bee bread. Oven drying has been proven to be the best method to preserve bee bread over frozen and refrigeration method based on the reduction in moisture content and microbial load .w suitable for microbial colonisation, which is 0.60–1.00. The preservation method such as drying until Aw goes below this level and can help prolong the bee bread shelf life. High Aw of fresh bee bread provides a favourable environment for the growth of beneficial bacteria, pathogenic bacteria, fungi, or mould [w parameter should be critically observed for quality control during storage.Bee bread has water activity between 0.60 and 0.92  within tor mould ,73,74. Tor mould . TherefoMelipona scutellaris (pH 3.28) [As previously mentioned, bee bread is more acidic than bee pollen. Duarte et al.′s  study onpH 3.28) . The pH pH 3.28)  suggestipH 3.28)  However,Aloe greatheadii var. davyana and Helianthus annuus bee bread in comparison to its fresh pollen.Tetragonula testaceitarsis bee bread had the lowest value with 43.1% [Tetragonula biroi Friese from the Philippines has the highest value with 59.94% [Most bee bread from Southeast Asia (SEA) is reported to have higher carbohydrate values than bee bread from other regions. Thailand′s th 43.1% , while Th 59.94%  among SETetragonula biroi from the Philippines [Meliponini subnitida from Brazil [Trigona spp. from Malaysia [Tetragonula biroi bee bread [The sugar analysis comparison  shows relippines , Meliponm Brazil ,87, and Malaysia . Other oee bread .Apis mellifera L. salivary (thoracic) gland secretes invertase, amylase, and glucosidase [Meliponini, Trigonini, and Scaptotrigona spp. could digest pollen polysaccharides into simple sugar using enzymes α and ß-amylase and α-glucosidase [Bee incorporates nectar and its salivary enzymes during pollen agglutination . Dependicosidase . Stinglecosidase . Simple Bee bread is rich in protein content, thus becoming the main protein source for bee development. The protein levels are varied across different geographical locations and between bee species (10.19–47.4%) . Pollen Tetragonula biroi from the Philippines and found mean values of only 1.83 g/100 g bee bread. Among the essential amino acids, leucine, and phenylalanine were highly present in the studied bee bread , and the most abundant saturated fatty acid was capric acid (1.89–5.66 g/100 g) and the common PUFAs were Omega-6 (α-linoleic acid) (0.50–1.63 g/100 g) and omega-3 (0.30–0.86 g/100 g) [Bee bread has diverse fatty acid profiles, which provides benefits to the bee nutrition and also for human health. According to Szczęsna , the mosg/100 g) .Tetragonula laeviceps bee bread. The ratio of polyunsaturated to saturated fatty acids was 1.59, higher than the ideal ratio of 1, which can slightly reduce HDL cholesterol level [Melipona mandacaia bee bread [trigona species using gas chromatography (GC-MS). Propanoic acid and palmitic acid were the most abundant saturated fatty acids in Trigona apicalis (4.04%) and Trigona thoracica (1.28%) bee bread, respectively. Meanwhile, minute amounts of omega-3 and omega-6 fatty acids were detected in range of (0.07–0.11%). In addition, analysis of volatile organic compounds (VOCs) found acetic acid as the sole organic acid in Australian Tetragonula carbonaria and Tetragonula hockingsi but absent in Austroplebeia australis [However, in another study, palmitic acid (1.65 g/100 g) was the prevalent fatty acid followed by omega-6 (1.52 g/100 g) in ol level . Similaree bread . Omar etee bread  analysedustralis .Phenolic compounds are secondary plant metabolites found widely in plants as a protective mechanism when encountering biotic or abiotic stress. They include phenolic acids, flavonoids, proanthocyanidins, and more. Intake of food containing phenolic compounds is sought to reduce the risk of gaining chronic diseases because of its antioxidant and anti-inflammatory properties . AccordiHeterotrigona itama bee bread yielded different amounts of polyphenols. Ethanolic extracts were found to have higher TPC (21.32–22.54 mg GAE/g) and TFC (16.48–26.57 mg QE/g) than aqueous extract [Austroplebeia spp. and Tetragonula spp. bee bread. [TPC and TFC content varies for different stingless bee species even from the same location . The sol extract . When coe bread. . HoweverTetragonula angustula bee bread. TPC ranged from 1053.1 to 2627.4 mg GAE/100 g pot-pollen, while the TFC was between 104.6 and 676.4 mg QE/100 g pot-pollen. In another study, Vit et al. [Frieseomelitta sp. and Melipona spp. with 1018.0–2085.0 mg GAE/100 g pot-pollen and 75.7–656.3 mg QE/100 g pot-pollen, respectively.In Southern Venezuela, Vit et al.  determint et al.  detectedApis spp. [pis spp. , fruits pis spp. ,103.Heterotrigona itama bee bread contains an average of 0.1108 mg vitamin C/g bee bread, which is considerably low based on the daily recommended intake [Analysis of bee bread vitamin content is still scarce. d intake . Becaused intake ,108. Extd intake .Tetragonula biroi [The most abundant mineral in stingless bee bee bread is potassium followed by phosphorus. From the studies conducted , potassila biroi .Heterotrigona itama [H. itama samples exceeding the proposed limit for heavy metal [Tetragonula biroi Friese bee bread but at a safe level [Apart from minerals, toxic metals such as arsenic, mercury, lead, and cadmium were detected in bee bread of na itama  and withvy metal . Belina-fe level . Toxic mfe level  as a resThere has been a tremendous report published on bee pollen as it is shown to possess therapeutic properties such as antioxidant, anti-inflammatory, antibacterial, anti-fungicidal, hepatoprotective, and anti-atherosclerotic activities . In contM. compressipes manaosensis inhibited P. aeruginosa, M. smegmatis, and Candida albicans efficiently and also mosquito larva C. quinquefasciatus, a vector for human parasitic worm Wuchereria bancrofti, in a concentration-dependent manner.Bee bread extract contains phenolic compounds, which can be attributed to its antimicrobial properties. Therefore, its importance as an antimicrobial agent has to be acknowledged not only towards bacteria but also towards yeast and parasite. Carneiro et al.  demonstrHeterotrigona itama bee bread against Bacillus cereus, Staphylococcus aureus, Escherichia coli, and Salmonella sp. Gram-positive bacteria were more sensitive towards the bee bread extracts, whereas ethanolic extract demonstrated stronger antibacterial activity compared to hexane extract.Akhir et al.  used ethAustroplebeia australis, Tetragonula carbonaria, and Tetragonula hockingsi in Australia. The extracts were effective against both Gram-positive  and Gram-negative bacteria . Similarly, extraction using ethanol showed better results as shown by the lowest minimum inhibitory concentration recorded by ethanolic extracts of Tetragonula hockingsi bee bread.Meanwhile, Perez-Perez et al.  examinedFrieseomelitta, Melipona, and Tetragonisca spp. from Venezuela and found that they exhibited antibacterial effects against Bacillus subtilis, Staphylococcus aureus, Enterobacter cloacae, and Pseudomonas aeruginosa but not against Escherichia coli. The antibacterial activities were correlated to the phenolic content.Sulbarán-Mora et al.  investigPhenolic compounds are one the most important natural antioxidants, which can be found in fruits, vegetables, tea, herbs, and essential oils. Antioxidant removes overproduced free radicals or reactive oxygen species (ROS), which cause molecular damages on DNA, protein, and lipid. Consequently, it has been linked with onset of diseases such as cancer and inflammation . StingleThere are multiple methods to assess the antioxidant activity of a plant extract and 2,2,-di-phenyl-2-picryl-hydrazyl (DPPH) is the most common in vitro method applied due to its simplicity, cost effectiveness, and rapidity . HoweverH. itama bee bread were determined using DPPH and 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) [Antioxidant activity is also affected by solvent extraction. For example, the antioxidant activities of ethanolic extract from ) (ABTS) . The ethAustroplebeia australis, Tetragonula carbonaria, and Tetragonula hockingsi bee bread when using ABTS, Fenton type reaction, and hydroxyl radical, respectively. The antioxidant activities also varied according to bee species. Comparison between trigona species showed Trigona thoracica bee bread with the highest DPPH inhibition (IC50/0.86 mg/mL) followed by Trigona apicalis (IC50/1.05 mg/mL) and Trigona itama (IC50/3.24 mg/mL) [However, Perez-Perez et al.  found si4 mg/mL) .Bee bread antioxidant ability corresponds to phenolic compounds ,107,119.Obesity is a public health concern. According to World Health Organisation , in 2016Heterotrigona itama bee bread reduced Lee obesity index and levels of total cholesterol (TC), low-density lipoprotein (LDL), fatty acid synthase (FAS) activity, atherogenic index, oxidised-LDL (oxLDL), and malondialdehyde (MDA) and significantly increased aortic antioxidant enzymes activities (superoxide dismutase (SOD) and glutathione peroxidase (GPx)) in high-fat diet (HFD)-induced obese rats. En face aorta images showed smaller adipocytes sizes and absence of atherosclerotic plaque in obese rats supplemented with bee bread [For example, ee bread .H. itama bee bread attenuated renal pathology caused by obesity by diminishing oxidative stress and downregulating the expressions of inflammatory markers and bax-mediated proapoptotic condition in the kidney of HFD obese rats.In another research, Eleazu et al.  studied Melipona fasciculata and Scaptotrigona affinis postica ethanolic bee bread extracts were administered in induced-oedema mice model in two separate studies [Some studies also suggest bee bread extract’s ability to reduce inflammation.  studies ,106. TheMelipona fasciculata and Scaptotrigona affinis postica. Upon investigation, Melipona fasciculata bee bread extract (500 mg/kg) reduced the abdominal contort in mice suffering from acetic acid exposure better than indomethacin. Meanwhile, the formalin test showed bee bread extract efficiency similar to those of indomethacin in reducing biting/licking time in mice [Bee bread extract has the ability to block pain detection through its antinociceptive activity. In vivo studies were conducted on pain-induced mice administered with bee bread ethanolic extract of two different species— in mice .Scaptotrigona affinis postica ethanolic extract at lower dosage (250 mg/kg). The bee bread treatment showed similar effect to indomethacin in the acetic acid writhing test but significantly better than the drug (p < 0.005) in the formalin test. The presence of polyphenols and flavonoids was suggested to be responsible for these activities [In a different study, Lopes et al.  investigtivities ,106.Bee pollen conversion into bee bread via lactic acid fermentation is linked with microbial action. Bee bread has been assumed to undergo microbial fermentation to increase its nutritional value ,48. ThisBifidobacterium, Bacillus, and yeasts from different bee bread species are summarised in In this section, understanding the bee bread microbial ecology will include data of bee bread from other bee species such as the honeybee and solitary bee due to limited record on stingless bee bee bread microbes. Beneficial bacteria isolated from bee bread, such as LAB, Aspergillus niger, Zygosaccharomyces rouxii, and Candida sp. [Bacillus spp. from Heterotrigona itama bee bread with enzyme and antimicrobial-producing properties. In addition, Mohammad et al. [Regardless, isolation and identification of microbes from bee bread have been widely reported. Yet, the study on their functional properties towards the bee ecosystem and most importantly, their potential for industrial application is still incomprehensive ,124. Somdida sp. . To datedida sp.  identifid et al.  isolatedLactic acid bacteria (LAB) such as Lactobacillus, Enterococcus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Carnobacterium, Aerococcus, Vagococcus, Oenococcus, Tetragenococcus, and Weisella  produce Apis mellifera species. Only a few studies have discussed on microorganisms in the stingless bee [Lactobacillus sp. particularly Lb. kunkeei is commonly found in bee bread [Apis mellifera belongs to Lactobacillus spp. which is largely composed of Lb. kunkeei [Lb. kunkeei was detected in low quantities in fresh bee bread of solitary bee Osmia cornuta and absent in the old bee bread of Osmia coruta and bee bread of H. itama [Current literature has conveyed extensive study conferring on microorganisms associated with honeybee bee bread or its ecosystem overall, particularly for less bee . The bacee bread ,129,130. kunkeei . HoweverH. itama ,130.Fructobacillus fructosus from H. itama bee bread. Fructobacillus spp. is a group of fructophilic LAB, which prefer fructose substrate over glucose [H. itama honey [Apis mellifera bee pollen, and bee bread [Recently, Mohammad et al.  isolated glucose  and can ma honey , honeybeee bread ,134 withApis without cultural bias. Oenococcus and Lactobacillus were the most active microbes in bee bread, comprising 52% and 60% of bacteria, respectively, suggesting its important contribution towards bee pollen conversion.Culture-independent studies help to provide more useful insight into the bee bread bacterial community, taking into account species which are unculturable under controlled condition. Mattila et al.  used 454Bacillus seem to hold its importance in the bee bread metabolic conversion. Although Gilliam [Bacillus in honeybee bee bread, the lactic acid production is less efficient than lactic acid bacteria fermentation. The functional role of Bacillus in bee bread was not defined until now. One of the early assumptions of Bacillus role in bee bread production was to produce enzymes for bee pollen conversion [Bacillus spp. such as B. subtilis [B. megaterium [B. licheniformis [Melipona fasciata [H. itama [Tetragonula biroi Friese [Apis mellifera [Osmia cornuta [Bacillus fermentation using bee pollen or bee bread is not yet reported.Lactic acid bacteria have been the integral focus when it comes to bee bread microbial fermentation, but other bacterial species such as  Gilliam  found Banversion . Few Bacsubtilis , B. megagaterium , and B. niformis  have exhfasciata , H. itamH. itama ,16, Tetri Friese , honeybeellifera , and sol cornuta . HoweverApis mellifera bee bread as opposed to bee pollen and flower pollen. Several studies had also attempted to describe the yeast communities not only in bee bread but also in bee colonies. In Brazil, yeasts such as Starmerella and Candida spp. are the most frequently found in the body, pollen, honey, and propolis of stingless bee species of Tetragonisca angustula, Melipona quadrifasciata, and Frieseomelitta varia [Starmerella spp. also has been highly associated with other stingless bee species. Teixeira et al. [Tetragonisca angustula′s pollen to harbour Starmerella meliponinorum. Meanwhile, Daniel et al. [Starmerella neotropicalis as the most abundant in Melipona quinquefasciata bee bread and bee pollen.Fermentation is commonly linked with yeast. Yeasts association with fermentation of bee bread has been proposed by researchers. An early study by Gilliam  discoverta varia . Starmera et al.  found Tel et al.  have chaLactobacillus and 3 Bifidobacterium strains were isolated from A. mellifera bee bread [Natural fermentation of bee bread is somehow related to bee gut microbiota . Understee bread , which aee bread .The earlApis mellifera bee gut were also found in fresh flower and nectar, suggesting common horizontal transmission [The scale of contribution of bee gut towards bee bread microbial inoculation has been studied by Mattila et al. . It was smission . NeverthApis spp. is widely discussed [Meliponini beecheii , M. bocandei (Africa), and Trigona sp. (Borneo and Thailand) revealed their honey stomach harbours Lactobacillus and Bifidobacterium species [Understanding bee gut microbiome could get complicated because of diverse bacterial communities . By far,iscussed ,149 whil species .Austroplebeia australis, Tetragonula carbonaria, and Tetragonula hockingsi). Interestingly, the microbiome of these stingless bee is different intercolonies but almost similar to the microbiome of honeybee Apis mellifera [Gilliamella, Bifidobacterium, Lactobacillus Firm-4 and Lactobacillus Firm-5 comprising the core of the gut’s microbiome, with Meliponini as the most diverse one [The gut microbiome not only varies between species but also between colonies. For example, Leonhardt and Kaltenpoth  focused ellifera . Comparierse one .A. mellifera gut microbiota through metagenomic analysis showed a possible link of these microbes to bee protection against pathogens and nutrition acquisition by degrading the pollen wall [Bee gut microbiota is maternally inherited , acquirelen wall . Zheng elen wall  found enApis mellifera to protect bee bread against bee pathogen M. plutonius in vivo and in vitro. However, these studies were heavily based upon honeybee Apis sp. More studies should be conducted on stingless bee, whether it will have similar outcomes or not.Understanding the functional role of bee gut, especially in bee bread fermentation, could be useful. According to Anderson et al. , fermentApis bee pollen in the current market and research focus. The food industry has seen tremendous changes in food trends from processed food to natural products with scientifically proven health benefits. The collective pieces of evidence in recent years have shown the potential of the stingless bee bee bread to be developed as a food ingredient, feed, or a supplement. It is rich in micronutrients, minerals, and phenolic compounds. However, research on their therapeutic values is still scarce. There are wide research opportunities to delve in on the bioactive compounds and their biological properties in vitro and in vivo. More research should be conducted so that an international standard for bee bread of stingless bee could be developed. More research on stingless bee bee bread can add to its commercial value and push to develop the meliponiculture industry. Bee bread has emerged as a reservoir to isolate beneficial microbes that contribute to bee bread preservation and can also provide newfound application in the industries.This review paper has summarised the physicochemical properties and health benefits of stingless bee bee bread and has discussed the microbial ecology of bee bread in general in relation to the bee gut. Bee bread is one of the overlooked stingless bee products, highly competing with


Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Somatic copy number alterations (CNAs) play a significant role in the development of this lethal cancer. In this study, we present a meta-analysis of CNAs for a total of 1,395 HNSCC samples. Publicly available R packages and in-house scripts were used for genomic array data processing, including normalization, segmentation and CNA calling. We detected 125 regions of significant gains or losses using GISTIC algorithm and found several potential driver genes in these regions. The incidence of chromothripsis in HNSCC was estimated to be 6%, and the chromosome pulverization hotspot regions were detected. We determined 323 genomic locations significantly enriched for breakpoints, which indicate HNSCC-specific genomic instability regions. Unsupervised clustering of genome-wide CNA data revealed a sub-cluster predominantly composed of nasopharynx tumors and presented a large proportion of HPV-positive samples. These results will facilitate the discovery of therapeutic candidates and extend our molecular understanding of HNSCC. Head and neck squamous cell carcinoma (HNSCC) has an incidence of over 600,000 new diagnoses per year worldwide, with a mortality rate of nearly 50% , 2. It cGenomic instability in the form of copy number alteration (CNA) is a hallmark of cancer cells and a promotor for carcinogenesis –8. CNAs Recently, a new phenomenon, called chromothripsis, has been described as a new mechanism for cancer initiation and progression . It is cHere we present a meta-analysis of genomic copy number alterations for 1,395 HNSCC samples from 19 datasets. In brief, we identified 72 and 53 regions of amplification and deletion, respectively, and detected potential driver genes in these regions. We identified 92 samples with the signs of chromothripsis, giving the incidence of 6%. Furthermore, we found some chromosome pulverization hotspots in HNSCC. The hierarchical clustering analysis identified two major clusters and cluster specific CNA patterns were provided. Our analysis may facilitate further studies on the distinct molecular mechanisms underlying HNSCC.We collected genomic array data from NCBI Gene Expression Omnibus (GEO)  and TCGAn = 768, 61%), nasopharynx , oropharynx , hypopharynx , larynx , and sinonasal cavity . The majority of patients were tobacco users (69.4%), while 55.6% were users of alcohol. Approximately 14%  of patients were HPV-positive which is consistent with the reported frequency at about 14.17%   . A summaFor GEO datasets, the raw signal intensity files were downloaded for re-analysis , 32. TheAffymetrix raw data files (.CEL) were re-analyzed by the R package aroma.affymetrix with the CRMAv.2 method . For nonq-value was set to <0.05 for peak detection, (2) the arm peel method was used to reduce noise, (3) the confidence level used to calculate the region containing a driver was set to 0.95, (4) “Extreme” method was applied for reducing marker-level to the gene-level copy number data, (5) log2 ratios >0.2 and <-0.2 were taken as thresholds for gain and loss detection. Furthermore, for known cancer-related genes, we downloaded 719 cancer consensus genes from the Catalog of Somatic Mutations in Cancer (COSMIC) database (GISTIC is a tool used for the detection of peak regions significantly amplified or deleted in a number of samples . Defaultdatabase .http://cgma.scu.edu.cn/CTLPScanner/) (We detected chromothripsis-like phenomenon by CTLPScanner web server (canner/) . The segThe genomic starts and ends of copy number alterations were considered as breakpoints. We used a stringent definition of CNA breakpoints to reduce the bias caused by technical or biological noise. A genomic position was defined as breakpoint if the Log2 signal value alteration between two adjacent genomic segments was >0.4 , 40. CopIn our collections, the genomic alterations ranged from whole arm gains and losses to focal high-level amplifications. The mean size of the CNAs was 6.7 Mb, and the average number of CNA events per tumor sample was 443. The CNA frequency plots for each chromosome are shown in r = −0.545, p = 0.0003) between arm-level gain and loss frequencies  (GISTIC 2.0  was perf < 0.05) . The ann < 0.05)  (Supplem < 0.05) . Among t < 0.05) , 51. Alt < 0.05) , 53. It  < 0.05) , 55. It  < 0.05) . These rp = 0.61)  .p < 0.01)  . Moreove < 0.01) . Among t < 0.01) . These rWe performed unsupervised hierarchical clustering on the copy number profiles of all HNSCC samples. CNA data were clustered in R based on Euclidean distance using Ward's method, resulting in two major clusters . Tumors The high degree of genomic heterogeneity of HNSCC underscores a challenge to distinguish between driver and passenger alterations from aberrant regions. A large number of tumor samples need to be examined in order to identify driver genes that are not detected when each tumor is analyzed individually. In this study, we characterized genome-wide DNA copy number alterations in HNSCC based on published high resolution aCGH and SNP array data. From the overall CNA profile of 1,395 HNSCC samples, we obtained the most frequent arm-level alterations. Previous studies have indicated that broad and focal copy number alterations may represent different characteristics of cancer cells with independent mechanisms , 59. WhiWe applied GISTIC algorithm  to perfoThe single catastrophic event chromothripsis is a novel phenomenon of genomic instability and is distinct from the progressive accumulation of mutations model of cancer development , 23. TheGenomic rearrangement breakpoints may disrupt tumor suppressor genes or create novel gene fusions with oncogenic potential. We performed genome-wide analysis and identified 323 recurrent breakpoints that were more clustered than would be expected if location of CNAs were randomly distributed throughout the genome. These breakpoint-prone regions revealed HNSCC specific genomic instability regions, by comparing with known common fragile sites and non-fragile regions of human genome. A number of potential driver genes detected by GISTIC were located in these regions. These results expanded our understanding of the impact of genomic rearrangements as a cause of HNSCC.We performed unsupervised hierarchical clustering analysis based on primary tumor samples from six different sites of head and neck region. The results did not show a clear separation between the samples from different locations, which demonstrated the diversity of genetic alterations in a variety of HNSCC sites, as well as the importance to collect large-scale datasets for analysis. In consistent with previous studies, we observed a sub-cluster with few CNAs, which may represent the M class cancer that predominantly driven by mutations rather than copy number alterations . In 2015In summary, we performed a comprehensive characterization of somatic genomic alterations based on a large cohort of HNSCC samples. Several newly identified driver genes in focal alterations, such as IRAK1BP1 and PTPN1, may facilitate molecular mechanism studies of HNSCC. Analysis of chromothripsis regions and breakpoint hotspots directed 8p and 11q as commonly affected regions in HNSCC that may related to disease development. Clustering of patients may provide evidence for patient stratification and differential treatment. These results provide valuable new insights into the mechanisms of genomic instability in HNSCC, and will facilitate the discovery of therapeutic and diagnostic candidates.https://portal.gdc.cancer.gov/) and NCBI GEO database (https://www.ncbi.nlm.nih.gov/geo/). We integrated the following datasets: TCGA-HNSCC, GSE11938, GSE20306, GSE20939, GSE23831, GSE25103, GSE31984, GSE33229, GSE33983, GSE34507, GSE36790, GSE39367, GSE40777,GSE47443, GSE51265, GSE57201, GSE66136, GSE68717, and GSE85514.Publicly available datasets were analyzed in this study. This data can be found in TCGA data portal (HC conceived the study. JY and YC collected raw data and performed data analysis. HL participated in interpretation of results and prepared figures. HC and JY drafted the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Microcystis sp. possessing a series of MC synthesis genes (mcyA-mcyJ) are well documented for their excessive abundance, numerous bloom occurrences and MC producing capacity. About 246 variants of MC which exert severe animal and human health hazards through the inhibition of protein phosphatases (PP1 and PP2A) have been characterized. To minimize and prevent MC health consequences, the World Health Organization proposed 1 µg/L MC guidelines for safe drinking water quality. Further the utilization of bacteria that represent a promising biological treatment approach to degrade and remove MC from water bodies without harming the environment has gained global attention. Thus the present review described toxic effects and bacterial degradation of MCs.Microcystins (MCs) classified as hepatotoxic and carcinogenic are the most commonly reported cyanobacterial toxins found in the environment.  Cyanobacteria are organisms that inhabit surface and bottom water. These organisms can accumulate to form blooms and scums which are mostly found on the water surface. WHO  reportedMicrocystis sp. owing to its extreme abundance, frequent bloom occurrences and ability to generate MCs MC-LR within 24 h  MC-LR . Novosphhin 24 h . Furtherectively . In a re 120 min . The eviAcinetobacter sp., Hyphomicrobium aestuarii, Pseudoxanthomonas sp., Rhizobium sp., Sphingobium sp., Sphingomonas sp. and Steroidobacter sp. from Taiwan reservoir, China [Acinetobacter sp., Aeromonas sp., Novosphingobium sp., Ochrobactrum sp., Pseudomonas sp., Rhodococcus sp., Sphingomonas sp., Sphingopyxis sp., Stenotrophomonas sp. and Steroidobacter sp. from drinking water reservoir in Southern California [Chryseobacterium sp. and Pseudomonas fragi was also found to completely degrade MC-LR [Agrobacterium sp., Bosea sp., Brevundimonas sp., Hyphomicrobium sp., Rasbo sp., Rhizobium sp., Rhodococcus sp., Roseomonas sp., Mesorhizobium sp., Nitrosococcus sp., Sandaracinobacter sp. and Sphingomonas sp. isolated from the mucilage of M. aeruginosa colonies during a bloom in a French pond completely degraded MC-LR and Des-MCLR. It is of interest that the bacterial community also degraded cyanobacterial secondary metabolites such as cyanopeptolins and aerucyclamides [The combination of two or more bacteria strains  have also proven to be capable of degrading MC-LR . The sevr, China  and 10 ilifornia  successflifornia . In a rede MC-LR . The natclamides . The finmlr gene cluster is fully described. A novel pathway involving four genes (mlrABCD), three intracellular hydrolytic enzymes (MlrABC) and two intermediate products; linearized MC-LR (Adda-Glu-Mdha-Ala-Leu-Masp-Arg-OH) and tetrapeptide (Adda-Glu-Mdha-Ala-OH) for MC-LR degradation using Sphingomonas sp. ACM- 3962 was demonstrated [mlrA gene cleaved the Adda-Arg peptide bond of MC-LR, which converted MC-LR to linearized MC-LR. The MlrB enzyme encoded by mlrB gene hydrolyzed the Ala–Leu bond, converting the linearized MC-LR to a tetrapeptide. The MlrC enzyme encoded by mlrC gene broke the tetrapeptide into smaller peptides and amino acids. Transport of MC and its degradation products were assumed to be facilitated via mlrD gene [Sphingomonas sp. B-9. Successive studies have also confirmed the existence of mlr cluster components in other MC-degrading bacteria species and strains  degradation with different intermediate products [Sphingopxyis sp. m6. These findings also suggest that more than three intermediate products are capable to occur during MC degradation. In a recent study Yang et al. [paa gene clusters for phenylacetic acid (PAA) degradation in the neighborhoods of mlrABCD gene cluster. Interestingly complete MC-LR degradation by Sphingopyxis sp. YF1 without Adda as the final degradation product (as illustrated in 2 through tricarboxylic acid (TCA) cycle [Regarding the sequential enzymatic hydrolyses of peptide bonds, lrB gene ,136. Sphproducts ,120,121.products  identifig et al.  found a A) cycle . The eviBordetella, Sphingomonas, Sphingopxyis Stentophomonas and Rhizobium [Acinetobacter sp., Hyphomicrobium aestuarii, Pseudoxanthomonas sp., Rhizobium sp., Sphingobium sp., Sphingomonas sp. and Steroidobacter sp.  and Chryseobacterium sp. and Pseudomonas fragi (Acclimatized-TSFU) [mlr homologues genes, and linearized MC, tetrapeptide and/or Adda were evident as intermediate products. At present only the mlrA gene has been observed in Bacillus sp. [Bacillus sp., Paucibacter sp., Pseudomonas sp. and Ralstonia solanacearum as well as bacterial communities including Klebsiella sp. and Stenotrophomonas sp. (YFMCD1), Alcaligenes faecalis and Stenotrophomonas acidaminiohila (YFMCD4) and Bordetella sp., Burkholderia sp., Cupriavidus sp., Methylotenera sp., Polaromonas sp., Polynucleobacter sp., Ralstonia sp. and Variovorax sp.   and Microbacterium sp. and Rhizobium sp.   ,130 are llus sp. ,84,86. Allus sp. ,74,87 deMicrocystis containing a series of MC synthesis genes. MCs inhibit protein phosphatases and constitute a natural health hazards in the environment. MCs are chemically stable in water and conventional water treatment approaches have failed to completely remove them. MC toxic effect led to the establishment of WHO’s 1 µg/L MC guidelines for safe drinking water quality. To provide safe drinking water, biological degradation is considered the most successful solution for MCs removal in the natural environment. Bacteria identification for different variants of MC degradation has been reported from various environmental habitats worldwide. The metabolism of these bacteria showed four genes (mlrABCD), three intracellular hydrolytic enzymes (MlrABC) and three degrading products . However studies about Sphingopxyis that resulted to a hydrolytic pathway via the discovery of eight new intermediate products, identification of different intermediate products from varying MC variants and the downstream MC degradation route by PAA metabolism which identified CO2 as the final degrading product have paved way for additional investigations on the biodegradation mechanism of MCs. Further studies need to explore the PAA metabolism on other MC degrading bacteria strains. Moreover, whether any degradation products exist between Adda and PAA, how the degrading product can be converted into PAA, and the functional genes as well as proteins involved in the metabolism process, need to be determined.In this current paper, toxic effects and bacterial degradation of MCs were reviewed. Studies indicate that the cyclic heptapeptide hepatotoxins MCs are primarily produced by species of


R) genes have been identified. More than 20 major clubroot (CR) disease-related loci have been identified in Brassica vegetables and several CR-resistant genes have been isolated by map-based cloning. Fusarium wilt resistant genes in Brassica vegetables have also been isolated. These isolated R genes encode the toll-interleukin-1 receptor/nucleotide-binding site/leucine-rice-repeat (TIR-NBS-LRR) protein. DNA markers that are linked with disease resistance allele have been successfully applied to improve disease resistance through marker-assisted selection (MAS). In this review, we focused on the recent status of identifying clubroot and Fusarium wilt R genes and the feasibility of using MAS for developing disease resistance cultivars in Brassica vegetables.The genus Brassica contains important vegetable crops, which serve as a source of oil seed, condiments, and forages. However, their production is hampered by various diseases such as clubroot and Fusarium wilt, especially in Brassica vegetables. Soil-borne diseases are difficult to manage by traditional methods. Host resistance is an important tool for minimizing disease and many types of resistance ( B. rapa includes leafy vegetables such as Chinese cabbage (var. pekinensis), pak choi (var. chinensis), and komatsuna (var. perviridis), root vegetables such as turnip (var. rapa), and oilseed (var. oleifera). B. oleracea comprises commercially important vegetable crops with morphological variations such as cabbage (var. capitata), broccoli , kale , kohlrabi (var. gongylodes), and cauliflower (var. botrytis). B. napus includes the oilseed crop, canola/rapeseed.The genus Brassica belongs to the family Brassicaceae (Cruciferae) containing 37 different species , transmembrane receptor-like kinases (RLKs), cytoplasmic kinases (CKs), and proteins with atypical molecular motifs [R genes is different between monocotyledonous and dicotyledonous genomes. TIR-NBS-LRR genes are mostly absent in monocotyledons, while TIR-NBS-LRR genes are present in dicotyledons and usually more abundant than CC-NBS-LRR genes [The first tier of plant immunity is called pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) ,5. Plantr motifs . The conRR genes . The R gRR genes ,15,16.R gene into a crop depends on the identification of a positive phenotype, the identification of genetic markers for marker-assisted selection (MAS) breeding, and understanding of how the novel resistance will behave under different genetic backgrounds and pathogenic pressures in the field. Clubroot and Fusarium wilt are considered as devastating diseases, and they cause a significant yield loss of Brassica vegetables for many years over the world. Some clubroot-resistant lines are susceptible to the Fusarium wilt and vice versa. In this review, we focus on recent knowledge about R genes of clubroot and Fusarium wilt as several important R genes/quantitative trait loci (QTL) against these pathogens have been identified in Brassica vegetables. In addition, MAS has been used to improve the disease resistance, and several cultivars with higher resistance in Brassica vegetables have recently been developed. We will introduce recent information about R genes and the prospect of their possible utilization for Brassica breeding.In a practical sense, the successful deployment of a novel Plasmodiophora brassicae Woronin and is recognized as a major devastating disease in Brassicaceae that poses an emerging threat to Brassica crop production [P. brassicae is a two-phase process (Clubroot is caused by the obligate parasite oduction . Clubroooduction . The inf process . The resFusarium oxysporum f. sp. conglutinans/rapae (Foc/For). Fusarium wilt disease was first reported in the USA, then in Japan and China, and has now been found almost all over the world [Foc, Fusarium oxysporum f. sp. conglutinans)/For, Fusarium oxysporum f. sp. rapae) usually invades plants through their young roots, but can also invade through wounds in older roots [forma specialis (f. sp.) of F. oxysporum can cause disease in Brassicaceae. Foc causes disease in B. oleracea and B. rapa and For is specific to B. rapa [Foc, race 1 and race 2, have been reported in the genus Brassica to date: race1 has been found worldwide and race 2 has only been found in USA and Russia [Yellowing or Fusarium wilt is caused by he world ,26. The er roots ,28. Thiser roots ,30. The er roots . The pater roots ,28 and r B. rapa . Only twd Russia .CR genes have been identified and mapped in B. rapa, B. oleracea, and other Brassica species [B. rapa, 18 major CR loci have been identified , Rcr9, and CRs on chromosome 8 [CR genes were identified through QTL mapping using a range of resistant sources based on molecular markers, genotyping-by-sequencing (GBS), or bulked segregant RNA sequencing (BSR-seq) strategies.Clubroot disease resistance has been extensively studies in the genus Brassica. Several  species . In B. rentified , Table 1mosome 1 , CRc andmosome 2 ,36, Crr3mosome 3 ,48,49,50mosome 5 , Crr4 onmosome 6 , Crr1  population [CRa was mapped on chromosome 3. A candidate gene of CRa has been identified, and it encodes a TIR-NBS-LRR protein [Crr1a and CRb genes have also been identified by map-based cloning [CRb was isolated independently of CRa, but they were identical genes [Crr1a encodes TIR-NBS-LRR [The first pulation  and a do protein . Crr1a a cloning ,46,53. Cal genes ,38. Crr1P. brassicae infection identified differentially expressed proteins (DEPs) between the susceptible and resistant lines [Recently, proteomics in Chinese cabbage during response to nt lines . Gene onnt lines .B. rapa, no major CR genes or lines with strong resistance have been identified in B. oleracea [B. oleracea. Genetic analysis of CR in B. oleracea was studied using diallel crossing methods or segregating populations. Only one major resistance gene, Rcr7, has been identified, and it might be located on chromosome 7 (LG 7) in cultivars, Tekila and Kilaherb of cabbage [B. oleracea  through BSR-Seq and further mapped with Kompetitive Allele Specific PCR (KASP) analysis in Brassica nigra lines PI 219,576 [Rcr6 was the first gene identified and mapped in the B genome of Brassica species. All of the CR genes found in the genus Brassica encode TIR-NBS-LRR proteins [A single dominant gene  219,576 . The autB. oleracea have been reported against Fusarium wilt [B. rapa and B. oleracea and has been studied extensively in recent years (Two types of resistance (Type A and Type B) in ium wilt . Type A ium wilt ,76,77. Tnt years , Table 4B. rapa, transcriptome analysis was performed using resistant and susceptible lines. The differentially expressed R genes were identified and seven dominant DNA markers at R genes were developed. Two dominant DNA markers on Bra012688 and Bra012689 were completely linked to the resistance phenotype by an inoculation test, indicating that these two genes are candidates for Fusarium wilt resistance genes in B. rapa  marker KBrS003O1N10 was developed [R genes in the target region, re-Bol037156 and re-Bol0371578, were predicted as resistance genes, and re-Bol037156 gene, which encodes a putative TIR-NBS-LRR type R protein, has highly similar sequences among the resistant lines [FocBo1 locus was identified on chromosome 7 and this locus was fine-mapped by using 139 recombinant F2 plants derived from resistant cabbage (AnjuP01) and susceptible broccoli (GCP04) DH lines [FocBo1 gene was shown by fine mapping to be an orthologous gene of Bra012688 in Chinese cabbage [Recently, Type A resistance has been mapped and molecular markers have been developed in oleracea ,30,32. Teveloped . One mineveloped . Later, nt lines . FocBo1 Foc infected plants in both resistant and susceptible cabbage cultivars was also investigated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) after the in-solution digestion of xylem sap proteins [Foc in cabbage roots were also analyzed [Foc inoculation [The proteome of xylem sap of the non-infected and proteins . Twenty-analyzed , where 8culation .MAS is an indirect selection process where a trait of interest is selected based on a marker  linked to that trait. Selecting individuals with disease resistance using MAS involves identifying a marker allele that is linked to disease resistance rather than to the level of disease resistance. There are several types of DNA markers that have been used to identify disease resistance genes ,78,82,84CR genes exhibit higher resistance to the disease [P. brassicae show variation, and different resistance sources from either B. rapa or B. oleracea vegetables were attained by P. brassicae. This suggests a serious risk that a resistance gene can be overcome by pathogen variants [P. brassicae population, and 61 of 106 P. brassicae population overcame the resistance in at least one of the seven CR cultivars [CR genes show different reactions against the variable virulence of P. brassicae [CR loci are less resistant than the homozygous state [B. rapa possesses several major CR loci 1 showed moderate resistance, whereas three minor QTLs without the major one showed distinct susceptibility [PbBo(Anju)1 plays a crucial role in the expression of clubroot resistance, and pyramiding minor CR genes are also essential for achieving higher resistance [P. brassicae [CR genes, CRb and PbBa8.1, were combined through MAS and CR homozygous lines in developed B. napus. The homozygous lines demonstrated a higher resistance than the heterozygous lines [The complexity of plant–pathogen interaction is a problematic in the case of CR breeding due to the appearance of multiple races of the pathogen . Combina disease ,67,86. Tvariants . For exaultivars . There arassicae ,63,66,74us state . B. rapa CR loci , which msistance ,52,85,88proteins ,89. In Be is low . This mi disease . Three Qentified . The acctibility . Later, us lines .The Type A resistance to Fusarium wilt disease controlled by a single dominant gene has been successfully mapped and molecular markers have been developed: SSR marker KBrS003O1N10 , InDel mB. rapa, a Fusarium wilt resistance gene is located on chromosome 3, and CRa/CRb, Crr3, and CRk are located near this Fusarium wilt resistance gene. The CRa/CRb gene is the closest, being approximately 2 Mb in physical distance to the Fusarium wilt resistance gene 4, nearby this Fusarium wilt resistance gene. However, these loci are not completely linked to each other [B. napus, the association between susceptibility to Fusarium wilt and clubroot resistance against pathotype 3 was found, and these two resistance genes are located about 10 cM apart [Breeding cultivars that have resistance to both clubroot and Fusarium wilt is desired. However, inoculation tests against multiple pathogens or multiple races are difficult to perform on the same individual plant. Thus, DNA marker-based selection is useful for the identification of plants that have one Fusarium wilt resistance gene and multiple clubroot resistance genes. Furthermore, it is necessary to confirm whether these resistance genes are linked. In nce gene . Since rch other ,83,84. TcM apart . HowevercM apart , suggestR genes through MAS. For the genetic accumulation of R genes corresponding to wide pathogenicity, MAS is indispensable because it allows a precise identification of how many R genes are involved in a cultivar, and can monitor the accumulation of R genes in the progeny in the breeding program. To increase the durability of resistant cultivars to a broader spectrum of pathogen races, the combination of different R genes into a single line will be indispensable.From the results from various researchers, it has been demonstrated that the DNA markers developed can select for the genes that are required for the acquisition of resistance, and these markers could be a powerful tool for resistance breeding in Brassica species. The novel breeding method developed can reinforce resistance by pyramiding R genes/QTLs have been identified in Brassica against the diseases and are being used for the improvement of resistance in cultivars. In case of clubroot disease, a total of 18 major CR loci have been identified in B. rapa, whereas only one major CR locus (Rcr7) and about 50 QTLs were detected in B. oleracea. Moreover, one locus (Pb-Bn1) on the A genome with more than 30 QTLs in B. napus and one locus (Rcr6) on the B genome in B. nigra were also identified. Several types of DNA markers that are linked with disease resistance allele have been developed, and they have been used for MAS. However, when there are several pathotypes, it is necessary to match effective R genes with a specific pathotypes and develop the DNA markers. The accumulation of CR genes corresponding to a wide pathogenicity will be important for breeding resistant cultivars.Brassica production is hampered by various diseases, especially clubroot and Fusarium wilt. Many types of Foc-1) was identified in B. rapa and B. oleracea, and several DNA markers have been developed.A single type A dominant locus (R genes found from both diseases mostly encode a putative TIR-NBS-LRR. Understanding how plants cope with exposure to multiple pathogens such as P. brassicae and Foc will be important in breeding cultivars with multiple disease resistance.


Encapsulation can lead to improved efficacy and safety of antifungal compounds. The attention of scientists has recently turned to biocompatible lipids as the carriers for the delivery of antifungal drugs, such as fluconazole. Although several research reports have already been published on fluconazole loaded solid lipid nanoparticles (FLZ-SLNs) and fluconazole loaded nanostructured lipid carriers (FLZ-NLCs), the possible advantages of NLCs over SLNs have not yet been fully established. Studies performed so far have given several contradictory results.Both formulations of fluconazole were synthesized using probe ultrasonication method and the characteristics were analyzed. Antifungal susceptibility testing (AFST) was performed with FLZ, FLZ-SLNs, and FLZ-NLCs using CLSI document M60 against some common fluconazole-resistant Candida species.P<0.05). However, three species groups were not statistically different in terms of the activity of FLZ-NLCs.A significant decrease was observed in minimum inhibitory concentration values when both formulations were applied. Nonetheless, FLZ-NLCs were significantly more effective (Candida species more effectively, as compared to FLZ-SLNs.Based on the obtained results, FLZ-NLCs could reverse the azole-resistance phenomenon in the most common  Several classes of antifungal drugs have been developed for the treatment of both systemic and superficial fungal infections , among wCandida strains . FLZ and FLZ-NLCs were dispensed into 96-well microdilution trays at a final concentration of 0.063–64 µg/ml and 0.1–8% (0.1–8 g/ml), respectively. Statistical analysisCandida isolates against FLZ-NLCs and FLZ-SLNs, and Kruskal–Wallis test was performed for three groups of Candida strains . P-value less than 0.05 was considered statistically significant. The obtained data were analyzed in SPSS software (version18).Data were expressed as mean±standard deviation. Analysis of variance (ANOVA) was used following Dunnett’s test. Mann-Whitney U test was applied to determine the effects of FLZ-NLCs and FLZ-SLNs on the reduction of MIC values and examine the differences between the MICs of resistant Characterization of solid lipid nanoparticles and nanostructured lipid carriersThe particle sizes, zeta potential, and entrapment efficiency of the developed SLNs and NLCs are depicted in Antifungal susceptibility testing C.albicans, C. glabrata and C. parapsilosis. However, the results as CLSI recommended were interpreted after 48 h of incubation at 35◦C. C. albicans, C. glabrata and C. parapsilosis after the application of both formulations (P< 0.05).Using FLZ-SLNs, the MIC50 drug concentration was obtained as 4 µg/ml, all C. albicans, C. parapsilosis and C. glabrata strains. Notably, MIC90 values were obtained as 0.125, 0.062, and 0.106 µg/ml for C.albicans, C. parapsilosis, and C. glabrata, respectively. For all three species, the MICs were significantly decreased with the application of FLZ-NLCs (P< 0.05). The comparison of the results obtained for three species groups revealed that the activity of FLZ-NLCs was not statistically significantly different (P>0.05). In general, FLZ-NLCs resulted in a significant decrease in MICs (P<0.05).Most strains visibly developed after 24 hours of incubation at 35°C using the CLSI document M27-A3 susceptibility testing methodology for FLZ-resistant strains of Candida species, such as C. krusei and newly described C. auris strains, are intrinsically resistant or less susceptible to several classes of antifungals, whereas others, including C. glabrata, develop acquired resistance following exposure to antifungal agents . Tglabrata . This obglabrata . AnotherCandida species more effectively, as compared to FLZ-SLNs. Nanostructured lipid carriers have been developed to overcome the pitfalls of SLNs. In NLCs, the combination of lipid with solid lipids results in a higher element drug loading which leads to a higher loading capacity for compounds. In addition, NLCs control the release of the drug and increase the chemical stability of the incorporated drugs [As the results of the current study indicated, FLZ-NLCs could reverse the azole-resistance phenomenon in the most common ed drugs , 27. As ed drugs , 10, 16.ed drugs . Gupta eed drugs . In the Candida species, as compared to FLZ-SLNs. This novel delivery system could face Candida strains that exhibit low/no susceptibility to the conventional formulation of FLZ. Based on the obtained results, these novel drug formulations may increase the bioavailability and dissolution rate of VRC to enhance the performance of the formulations.Fluconazole solid lipid nanoparticles were found to be more effective against M. M, HR. K and M. S. designed the project and interpreted the data. AH. D. and M. N. performed the tests and revised and wrote the manuscript. In addition, all of the authors approved the final version of the manuscript.The authors declare that they have no conflict of interest regarding the publication of this paper.This research was financially supported by a grant (No957487) given to Maryam Moazeni by the National Institute for Medical Research Development (NIMAD). The funder had no role in study design, data collection and interpretation, or the decision to submit the work for publication.


Mahonia oiwakensis is a frequently-used traditional Chinese medicinal plant with efficient anti-tumor and anti-inflammatory ability. In this study, we assembled the complete chloroplast (cp) genome of M. oiwakensis. The complete cp genome of M. oiwakensis is 165,126 bp in length, and has a typical structure with large  and small  single-copy regions separated by a pair of inverted repeats  of large size. The M. oiwakensis cp genome contains 148 genes, of which 103 protein-coding genes, 37 tRNA genes, 8 rRNA genes. Phylogenetic analysis shows that M. oiwakensis closely clustered with Mahonia bealei, but were nested among three speices of Berberis, which robustly supports that Mahonia is not monophyletic, thus needs redefinition. Mahonia Nuttall is a taxonomical complex woody genus in Berberidaceae. Some taxonomic treatments  with the simple-leaved group (true Berberis). Phylogenetic analyses of the chloroplast and nuclear gene implied that Mahonia is the non-monophyly  is one of traditional Chinese medicinal plants that has been demonstrated to exhibit antioxidant, analgesic, anti-inflammatory and hepatoprotective effects , Bijie City, Guizhou Province, China and voucher specimens (DTH2017110806) were deposited at BJ . Total genomic DNA was extracted from the silica-dried leaves using the TIANGEN plant genomic DNA extraction kit, following the manufacturer’s instructions. The genomic paired-end (PE150) sequencing was performed on an Illumina Hiseq 2000 instrument . The complete cp genome was assembled using SOAPdenovo2  using Geneious Prime 2020.0.3. Annotation was performed via Geneious Prime 2020.0.3, coupled with manual check and adjustment.The mature leaves of M. oiwakensis is 165,126 bp in length, including two single copy regions  and two inverted repeat regions . The complete chloroplast genome sequence of the M. oiwakensis contains a pair of especially large IRs that was also found in M. bealei . The sequences were aligned using MAFFT v7 (Katoh et al. Berberis , which r