Deficiency in cerebral amyloid β-protein (Aβ) clearance is implicated in the pathogenesis of the common late-onset forms of Alzheimer’s disease (AD). Accumulation of misfolded Aβ in the brain is believed to be a net result of imbalance between its production and removal. This in turn may trigger neuroinflammation, progressive synaptic loss, and ultimately cognitive decline. Clearance of cerebral Aβ is a complex process mediated by various systems and cell types, including vascular transport across the blood–brain barrier, glymphatic drainage, and engulfment and degradation by resident microglia and infiltrating innate immune cells. Recent studies have highlighted a new, unexpected role for peripheral monocytes and macrophages in restricting cerebral Aβ fibrils, and possibly soluble oligomers. In AD transgenic (ADtg) mice, monocyte ablation or inhibition of their migration into the brain exacerbated Aβ pathology, while blood enrichment with monocytes and their increased recruitment to plaque lesion sites greatly diminished Aβ burden. Profound neuroprotective effects in ADtg mice were further achieved through increased cerebral recruitment of myelomonocytes overexpressing Aβ-degrading enzymes. This review summarizes the literature on cellular and molecular mechanisms of cerebral Aβ clearance with an emphasis on the role of peripheral monocytes and macrophages in Aβ removal. Alzheimer’s disease (AD) is a severe neurodegenerative disorder and the most common form of senile dementia, affecting over 5 million in the United States and 45 million worldwide , 2. AD mOver the past century, our understanding of the molecular mechanisms underlying the development of AD has greatly expanded. Though still pathological hallmarks, extracellular plaques and intracellular neurofibrillary tangles (NFTs) within the brain , compris40) and Aβ1-42 (Aβ42) alloforms are generated through a sequential cleavage of APP by a β-secretase (BACE1) and a γ-secretase transmembrane complex. Mutations within the gene encoding APP and its Aβ coding sequence were found to cause early-onset, autosomal-dominant inherited forms of familial AD (FAD) [APP gene develop AD-like Aβ and tau neuropathology, leading to cognitive decline [42 alloforms [APP mutation in non-demented Icelanders [APP  was shown to reduce amyloidogenic Aβ production and aggregation, providing protection against age-associated cognitive decline [Aβ denotes a group of endogenous peptides, typically of 36–43 amino acids. It derives from a larger transmembrane protein, the amyloid precursor protein (APP), in a complex proteolytic process, described extensively elsewhere . The disAD (FAD) . Similar decline . In addi decline –22 , and are termed sporadic or late-onset AD (LOAD). The etiology of LOAD is multifactorial: multiple genetic and environmental factors likely contribute to the development of disease. Strong support for the role of Aβ accumulation in both AD forms came from several clinical studies. While in FAD cases cerebral Aβ increase was explained by Aβoduction , deficiepatients . Despite42 were shown to be especially neurotoxic [Growing evidence indicates that Aβ exerts its neurotoxic effects in both an alloform- and conformation-dependent manner . Small, urotoxic –113 and urotoxic , 115. Spurotoxic –119. Addurotoxic  and accuurotoxic , disrupturotoxic . Aβ fibrurotoxic , 124 andurotoxic –127. Thiurotoxic . Beyond urotoxic . This phurotoxic . In muriurotoxic .in vitro and in vivo [42, preserved synapses and neuronal function as well as prevented cognitive decline [Although the existence of Aβ plaques and NFTs establishes the definitive diagnosis of AD, many researches have challenged the predominant belief that Aβ is central to the development of disease. For example, studies have demonstrated that NFT pathology correlates more strongly than amyloid plaque load with brain atrophy and cognitive decline , 133. In in vivo –143. Fur decline , 144–146 decline . Taken tIn this review, we summarize various cellular and molecular, physiologic mechanisms of Aβ removal from the brain. Specifically, we cover Aβ transport across the blood–brain barrier (BBB), glymphatic clearance, cellular uptake, and enzymatic degradation. Large-scale genetic studies have further cemented the connection between Aβ accumulation, clearance by innate immune cells, and disease risk, and will be the topic of the following section. Finally, we place a particular emphasis on the growing evidence supporting a key role for microglia, and moreover, monocyte-derived macrophages in the physiological clearance of cerebral Aβ see Fig. , and we APP, PS1, or PS2 leading to the amyloidogenic processing of APP and overproduction of synaptotoxic Aβ42 , complement activation , the HLA gene complex , and myeloid cell-mediated Aβ proteolysis  and phagocytosis  [CD33  and TREM2 (triggering receptor on myeloid cells 2) directly link impaired microglial and macrophage phagocytosis of Aβ to increased susceptibility to AD. The reported effect size of TREM2 variants on AD risk has varied in the literature [Historically, the study of AD-related genes pertained to the rare, inheritable and early onset forms of the disease (termed FAD) , 22. Theoping AD , 151. Apoping AD , 152–154oping AD , 96, 155oping AD . More sp, TREM2) , 156. Interature . Nonetheterature , 158. YeThe key mechanisms of Aβ clearance were shown to involve either Aβ removal to the peripheral blood and lymphatic systems or degradation within the CNS tissues. Aβ reaches the peripheral circulation via chaperone-mediated transport across the blood brain barrier (BBB) , perivasSecreted peptidases are critical for the catabolism of Aβ peptides. These enzymes were reported to have an affinity for specific domains within the Aβ amino acid sequence and an ability to cleave and convert these peptides to shorter, more benign forms –167. Tab42 into the less toxic Aβ40 alloform [ACE1 that reduces plasma ACE levels and increases risk for AD [42 levels were elevated after 11 months of treatment [ACE is a zinc-dependent peptidase with significant expression by endothelium throughout the body as well as by cortical neurons in the brain . Most wealloform . In postalloform , 207. Fualloform . It was k for AD –47. Howek for AD . Interesk for AD , suggestreatment . It is ireatment . In ADtgreatment , 191, de40 and Aβ42 into non-toxic fragments [APOE status [Apoε4/4 carriers, who exhibit reduced IDE expression.IDE is a zinc metalloprotease that is capable of degrading soluble Aβragments , 174. Alragments , 185 or ragments , where iragments , 210. Inragments –213, braragments –213, disragments , 213, anE status . In gene42 and Aβ40 [+/CD45hi monocytes  family. In general, MMPs are responsible for the degradation and maintenance of the extracellular matrix. MMP-9 has been shown to degrade compact plaques , 187 as and Aβ40 . In the and Aβ40 , microgland Aβ40 , astrocyand Aβ40 , and inf42 and Aβ40 [NEP is a type II integral membrane zinc metalloprotein with the bulk of its structure, including the active site, facing the extracellular space. NEP is expressed throughout the brain, predominantly on pre- and post-synaptic neuronal membranes , 169, anand Aβ40 , 172 butand Aβ40 , which mand Aβ40 , 224 or and Aβ40  in ADtg and Aβ40 , 172.in vitro and correlated with enhanced clearance of soluble Aβ42 [NEP, IDE, ACE and MMP-9 are Aβ-degrading enzymes expressed by innate immune cells and represent a crucial pathway by which these cells may eradicate pathogenic Aβ Table . Expressble Aβ42 .SWE/PS1ΔE9 mice infused with CD115+ monocytes or immunized with the altered myelin-derived antigens, such as glatiramer acetate (GA) or myelin oligodendrocyte glycoprotein-derived peptide (MOG45D) displayed increased accumulation of MMP-9-secreting macrophages surrounding Aβ plaques —prevent intracellular protein aggregation, and are thus instrumental in protecting against the neurotoxicity of cytosolic Aβ accumulation. In AD brains, however, these systems are considerably compromised –231. DegThe lysosome is the final destination for both autophagic vacuoles and the endosomes formed by receptor-mediated endocytosis. The latter process occurs in neurons and glia through a distinct set of molecular chaperones, discussed in greater detail in the following sections. Each lysosome contains a cocktail of hydrolytic enzymes capable of degrading Aβ; however, the hydrolytic machinery is often overwhelmed in AD , 232–23442 showed incomplete intracellular degradation, with non-degraded fibrils remaining in phagosomes for up to 20 days [Myeloid cells in particular may suffer AD-associated deficits in endosomal-lysosomal trafficking and Aβ processing. Microglia isolated from plaque-bearing sections of human AD tissue indicated that Aβ fibrils were located in the endoplasmic reticulum and deep invaginations of the cell membrane, instead of within endosomes or lysosomes . Even no 20 days , 240. Th 20 days . One pos 20 days .While healthy peripheral macrophages appear better equipped to degrade fibrillar Aβ than resident microglia, monocytes in AD patients exhibit lysosomal dysfunction , 229. SpThree types of autophagy exist: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). While both macroautophagy and CMA dysfunction are implicated in AD , 245, thThe UPS is a highly regulated degradation process for cytosolic short-lived and misfolded proteins. As such, it is an important protective mechanism against neurotoxic protein aggregates. Briefly, specific proteins are polyubiquinated by a series of ligases  for recognition and degradation by the 26S proteasome complex. Whether UPS dysfunction is a cause or consequence of AD-related degeneration remains unknown. In favor of the former, both ubiquitin conjugation and proteasome activity decline with age and in AD tissue , 261. ArRemoval of Aβ into the peripheral circulation is thought to facilitate the majority of physiologic Aβ clearance . TranspoLocated on the abluminal surface of brain endothelial cells, LRP-1 binds either ApoE-Aβ complexes or Aβ alone , 265, suPICALM is expressed on endothelial cells, and to a lesser extent, on neurons . PICALM In vitro studies have helped to elucidate the role of ApoE, demonstrating that it binds Aβ directly under certain conditions [Under physiologic conditions, ApoE is a carrier protein that maintains cholesterol and phospholipid homeostasis . Major Anditions . It is tnditions , 68. Supnditions . In contnditions , 284. Tanditions .APOE2 allele may be protective against AD [APOE4 alleles significantly increases the risk of developing AD and reduces the age of onset [APOE4/4 genotype is associated with accelerated and more pronounced cerebral amyloid pathology and CSF abnormalities [Three isoforms of ApoE exist in humans: Apoε2, Apoε3, and Apoε4 . Evidencainst AD ; conversof onset , 282. Fumalities . Severalmalities , 53, 152malities . Additiomalities , 287. Apmalities . In non-malities . A comprmalities , 288. Thmalities , 289.APOE4-targeted replacement mice with the 5XFAD ADtg model greatly increased microgliosis and astrogliosis surrounding Aβ plaques [Apoε4 may hinder other important mechanisms of Aβ clearance, namely, intracellular catabolism by neurons and innate immune cells. Specifically, Apoε4 may interfere with these processes by inducing lysosomal leakage or by impeding myeloid cell-mediated clearance , 236. Th plaques . Similar plaques . Further plaques . It remaThe glymphatic system is a pathway of brain-wide waste clearance for small proteins and metabolites. In this pathway, CSF enters the periarterial space and, driven by arterial pulsations, enters the brain parenchyma to exchange with the interstitial fluid (ISF). Bulk flow of CSF/ISF, containing extracellular molecules such as Aβ, are then driven to perivenous spaces for recirculation in the CSF or clearance to peripheral lymphatics , 162. GlWater channels known as aquaporin 4 (AQP4) are the key elements in CSF-ISF exchange, and thus clearance through the glymphatic pathway. AQP4 is located on astrocytic end feet and encircles the vasculature. Mice lacking astrocytic AQP4 showed reduced CSF influx by ~70% and decreased interstitial Aβ clearance by ~55–65% , 162. AdA growing body of evidence supports the emerging concept that activated inflammatory cells, mainly brain-resident microglia and infiltrating blood-borne monocyte-derived macrophages, are critical for the physiological clearance of Aβ , 295–298Today’s newer methodologies delineate subtle differences in marker expression, allowing for a more accurate categorization and attribution of function to these cell populations. For example, standard CD11b (MAC1), isolectin B4 (IB4), F4/80, or ionized calcium binding adapter molecule 1 (Iba1) markers in the brain are indistinguishably expressed by both infiltrating monocytes and resident microglia , 303. YeOther key developmental and functional differences between microglia and macrophages help distinguish these unique cell types. Microglia originate from hematopoietic stem cells of the yolk sac , while iHeterogeneous populations of these immune cells exist in the brain, especially in the diseased state. Their demonstrated clearance capacity varies given the experimental paradigm and the phase of disease studied. Table in vitro [in vivo clearance capacity has been questioned [Microglia aid in the normal development, function, and repair of the CNS. In response to injury or other pathological conditions, microglial processes and cell bodies migrate to lesion sites and initiate an immune response to contain and resolve particular insults , 299. Acin vitro , 332–334estioned –337. Sucestioned , 339, whestioned , 335–337estioned . Moreoveestioned –342. Eleestioned , 344 andestioned . Additioestioned , and canestioned , 347. Thin vivo two photon imaging also demonstrated that early on, microglia form a protective barrier around developing plaques, preventing accumulation of Aβ42 protofibrils and associated local neuritic damage [Although this indicates a detrimental role for microglia in the AD brain, plaque-associated microglia have been shown to degrade scar tissue proteins with secreted proteases, clear cellular debris, and recruit the adaptive arm of the immune system to stimulate or regulate effective local immune responses , 348. A c damage . Remarkac damage . A 1-houc damage . Currentc damage . Howeverc damage , 349. TaCD33 and TREM2, two genes negatively impacting the phagocytic and Aβ clearance capacity of monocytes, confer a greater risk of developing AD  is one of the principal receptors involved in Aβ uptake by immune cells. It is expressed on human and rodent macrophages , microglin vitro , 365 andarations , 366. Glarations , 367, waarations . Immunizin vivo. SCARA1-deficient APPSWE/PS1ΔE9 transgenic mice exhibited increased mortality and a significant elevation in surface area fraction stained for Aβ compared to control ADtg mice [The importance of SCARA1 function in Aβ clearance has also been established Dtg mice . IncreasDtg mice , 369 as Dtg mice . SCARA1 42 through interactions with two distinct receptor complexes acting as a functional unit [42 in microglia cell lines [42 directly [42 clearance [42 with continued expression of other scavenger receptors [CD36 is a type B scavenger receptor expressed on the cell surface of monocytes, macrophages, astrocytes, and neurons . CD36 hanal unit , 371. CDnal unit , and antll lines . Like SCll lines  . Thus, microglia may contribute to the toxic environment that induces their own impairment , 374. MoThe triggering receptor expressed on myeloid cells 2 protein is a single-pass type 1 transmembrane protein that is part of the immunoglobulin superfamily. Ligands of this receptor include anionic carbohydrates, phospholipids, and apolipoproteins such as ApoE –377. TRETREM2 as an AD risk factor in multiple populations [TREM2 risk alleles have also been identified, including R62H and D87N [in vivo [GWASs have recently implicated the R47H variant of ulations , 155. Inulations . Other Tand D87N , 155. Reand D87N . It was [in vivo . MoreoveTREM2 knockout in APP/PS1 mice greatly ameliorated disease progression [42 compared to wildtype cells, an impairment partially rescued by therapeutic anti-Aβ antibodies. Antibody-coated Aβ greatly enhanced phagocytosis by both TREM2 knockouts and wildtype cells, although clearance by mutant cells lagged behind controls under all conditions [TREM2 mutations, yet further research is needed to fully elucidate these relationships.Studies utilizing ADtg mouse models, however, point to a much more complex role for TREM2 than previously thought , 376. Ingression , while tgression , 376. Thnditions . This fi42 levels and plaque burden [42 is thought to explain this relationship. In support of this, possession of the newly discovered rs3865444C risk allele [42. Monocytes isolated from young individuals with the rs3865444C risk allele also displayed an Aβ42 phagocytic deficit [A allele dampens CD33 expression and increases the proportion of CD33 molecules that lack a SIGLEC-specific region responsible for phagocytosis inhibition [CD33 is a member of the sialic acid-binding immunoglobulin-like lectins (SIGLECS) family, expressed on myeloid cells , 379. Ine burden . The dimk allele  results  deficit . Enriche deficit . ConversDespite the surging data favoring a critical role of monocytes in AD pathophysiology, it is important to acknowledge the contradictory evidence in the field surrounding monocyte-mediated Aβ clearance in chronic neurodegenerative diseases. Major questions remain. (1) Under what conditions do monocytes infiltrate the CNS? (2) Do monocytes and macrophages behave differently from microglia once in the CNS parenchyma, especially in their ability to resist misfolded Aβ forms? (3) Is the neuroprotection exhibited by monocytes a predominantly peripheral blood or a local effect? And (4) Is the effect cell-mediated, molecular or plasma-mediated, or both? The following sections address these controversies given the available literature and identify methodological discrepancies that may have generated some confusion.Monocyte infiltration in AD was first documented by seminal studies transplanting GFP-labeled bone marrow cells into irradiated ADtg mice –297, 318+ or CD115+ monocytes from the bone marrow of young adult wildtype mice, rather than whole blood marrow, eliminating the additional confounder of increased progenitor cell numbers seen in earlier studies. Importantly, blood enrichment with GFP monocytes in age-matched wildtype (non-ADtg) animals did not cause recruitment of monocytes to the CNS [42 forms [To further elucidate the effects of irradiation, the GFP-transplantation paradigm was repeated, this time shielding the heads of recipient mice to conserve BBB integrity. This procedure reduced monocyte infiltration into the CNS, and called into question the conditions necessary for monocyte recruitment . However the CNS , 146, im42 forms , 383, an42 forms , 385.The mechanism by which cerebral amyloid accumulation induces monocyte infiltration is multifactorial. Vascular Aβ deposition can directly damage the vessel wall  and alloin vitro data [Modulation of monocyte recruitment to the CNS clearly demonstrates the significant contribution of monocyte-derived macrophages to Aβ clearance. Blocking CCR2 signaling , 331 or tro data , 241 ledtro data , 390.CD11b-HSVTK model, in which the thymidine kinase of the herpes simplex virus is expressed under the CD11b promoter, allows for elimination of local, proliferating myeloid cells upon intracerebroventricular administration of ganciclovir. Peripheral GFP-labeled macrophages can then repopulate the CNS, introduced by either transplantation [Other studies utilizing microglial ablation techniques challenge this assumption. Crossing ADtg mice with the antation  or parabantation . In bothantation , 354. Giantation , 391. Spantation , 392. InIt is undeniable though that certain conditions do in fact enhance the migratory and Aβ clearing capacity of infiltrating monocytes over their resident counterparts. In particular, ADtg mice immunized with the myelin-derived peptides MOG45D or GA exhibited reduced Aβ levels and neuroinflammation, attributable to the increased recruitment of anti-inflammatory monocytes that directly engulfed Aβ , 148. OthiCX3CR1intCCR2+) type pertaining to CNS recruitment and parenchymal Aβ clearance, and a patrolling (Ly6CloCx3CR1highCCR2−) type that remains associated with the vasculature [in vivo imaging of APP/PS1 mice has elegantly demonstrated that patrolling monocytes are in fact attracted to and crawl along Aβ-positive veins, where they engulf Aβ and subsequently recirculate into the bloodstream [Recognition of the heterogeneity of different monocyte subtypes has emerged from recent studies that identified new functional biomarkers for myelomonocytic cells. An immunohistochemical and activity-based distinction has been proposed between murine monocyte subsets: an inflammatory  or with GA also had profound effects on the function of innate immune cells, which consequently reduced various pathological features of AD. In ADtg mice, MOG45D-DC immunization increased CNS recruitment of anti-inflammatory macrophages, demonstrated by reduced TNF-α and increased IL-10 and TGF-β expression, that efficiently phagocytosed Aβ . As a re effects , 348, wh effects . In agre effects  or of TG effects , enhance effects , 400.+ monocytes to the peripheral blood of ADtg mice stimulated their spontaneous migration to amyloid lesions in the absence of irradiation, genetic manipulation, or chemotherapy. Treated mice exhibited reduced cerebral Aβ protein levels and astrogliosis, preserved pre-synaptic integrity, and ameliorated cognitive deficits [Other methods of immune modulation include adoptive transfer of healthy monocytes and bone marrow transplantation. Infusion of wildtype CD115deficits . Likewisdeficits , 318.42, limited plaques and astrogliosis, and preserved cognitive function [Because peripheral monocytes were shown to cross the BBB and home to sites of Aβ accumulation, they can function as a delivery system of therapeutic agents. Targeted overexpression of either NEP or ACE has proven beneficial in abrogating AD progression in murine models. Injecting 9-month-old ADtg mice with NEP-expressing monocytes completely prevented further Aβ deposition when compared to untreated ADtg mice or those infused with monocytes containing inactive NEP . Similarfunction , 191.Aβ clearance is a complex, multifactorial process, requiring the collaboration of various systems and cell types. Aβ can be removed to the peripheral circulatory or lymphatic systems by transport across the BBB or by absorption from the CSF and ISF. While innate immune cells are known to phagocytose and degrade fibrillar Aβ, these cells were only recently shown to engulf and clear soluble Aβ species as well. It is still unclear whether Aβ accumulation is a cause or consequence of disease. However, mounting evidence has shown that increased cerebral Aβ burden is the earliest pathognomonic event in AD. Moreover, soluble, oligomeric Aβ was shown to directly incite nerve and synaptic damage, leading to impaired neuronal function. In the late-onset, common cases of AD, Aβ buildup is attributed to defective clearance, rather than to its overproduction. The observed deficiency could result from impairments in any one of the removal processes or, more likely, a combination of minor clearance deficits and compounding risk factors that varies from patient to patient. Modulation of clearance mechanisms may be an important early strategy for curtailing Aβ accumulation and disease progression.CD33 gene impacts the phagocytic capacity of monocytes isolated from young adult patients, indicating that this particular functional deficit is present throughout life. Other GWAS data have linked multiple immune-related risk factors to AD. Known relationships between the major risk gene TREM2 and monocyte/microglia phagocytic function offer a compelling demonstration of the immune system’s impact in AD.As our knowledge of AD continues to expand, so does a body of evidence that supports a key role for innate immune cells, especially monocyte-derived macrophages, in Aβ removal, local immune regulation, and repair. Bone marrow-derived monocytes can cross the BBB and clear Aβ through cellular uptake and enzymatic degradation, perhaps even more efficiently than resident microglia. The clearance process is, again, complex. Phagocytosis requires the coordination of many surface receptors  for recognition and uptake, followed by intracellular trafficking, ultimately to lysosomes, for degradation. Monocytes, macrophages, and microglia also mediate extracellular Aβ degradation through surface expression or release of various proteases, such as ACE, IDE, NEP, and MMP-9. These functions were reported to be markedly impaired in peripheral monocytes isolated from AD patients. It is possible that the observed deficiency is a consequence of immune senescence and AD-related degeneration, or perhaps their dysfunction is a direct contributor to disease development. In support of the latter, possession of a rare variant of the AD-associated Aggregates of misfolded Aβ are known to trigger a prolonged neuroinflammatory response that is tightly associated with synaptic dysfunction and cognitive decline. Enhancing cerebral recruitment of monocytes through either peripheral infusion or immunization with altered myelin-derived antigens was shown to temper these degenerative changes in murine models. Specifically, monocytes were able to efficiently clear Aβ and resolve the resulting astrogliosis and neuroinflammation, thereby preserving synaptic integrity and cognitive function. Immunomodulation approachs that enhance cerebral recruitment of neuroprotective monocytes hold great promise as disease-modifying therapeutic interventions and represent a valuable target for further application and translation.


Myelosuppression is a common side effect in cancer patients receiving chemotherapy or radiotherapy. Chinese herbal medicine (CHM) has shown promise in alleviating myelosuppression. We searched for randomized controlled trials (RCTs) from seven databases without language restriction. We included RCTs in adults, in which hematological toxicity was measured according to WHO criteria and control group underwent chemotherapy and/or radiotherapy and the treatment group was given oral CHM. We searched 1021 articles from the date of databases inception to October 7, 2016. We selected 14 articles for the final analysis. Pooled data showed that CHM significantly decreased the suppression rate of leukocytes, neutrophils, hemoglobin, and platelets compared with the control group, particularly in grade III-IV toxicity . CHM as an adjuvant can alleviate myelosuppression induced by chemotherapy or radiotherapy, reduce grade III-IV toxicity, and maintain therapeutic dose and treatment cycle. However, due to heterogeneity and publication bias, the results should be interpreted with caution and validated by conducting strictly designed multicenter RCTs of high quality and large scale. Myelosuppression, also known as bone marrow (BM) suppression or myelotoxicity, is the major side effect of chemotherapy and radiotherapy. Blood originates in the BM  and blooIt is vital to improve survival rate of cancer patients being treated for anemia, infection, and other complications. However, some treatments could cause adverse effects. For example, G-CSF causes shock or chronic fibrous pneumonia and repetitive platelet transfusion leads to formation of alloantibody . TherefoStudies on CHM preventing myelosuppression have been inconclusive or conflicting. Currently, it is difficult to make the clinical decision of administering CHM as an adjuvant in chemotherapy or radiotherapy. A similar systematic review was published , which rhttps://doi.org/10.1155/2017/3432750).In this study, our objective was to minimize clinical heterogeneity and pool high-quality studies to generate robust evidence regarding the potential therapeutic value of CHM in preventing myelosuppression. This meta-analysis was reported and performed in accordance with PRISMA guidelines  and in which patients were over 18 years of age, control group was treated with radiotherapy or drug therapy and treatment group was treated with CHM along with radiotherapy or drug therapy, and hematological toxicity was measured mainly according to World Health Organization (WHO) criteria  Table 1Table 1. ∗”).We systematically searched databases, including China Academic Journal Network Publishing Database (CAJD), China Biology Medicine disc (CBMdisc), China Doctoral Dissertations Full-text Database (CDFD), China Master's Dissertations Full-text Database (CMFD), PubMed, Cochrane, and EMBASE, for relevant articles published from the date of the databases inception to October 7, 2016. There were no language restrictions. As part of search strategy, we used a combination of the following terms: “Chinese herbal medicine” ,” “Chinese herbal medicine,” “Chinese herbal drug,” “traditional herbal medicine,” “herbal medicine,” “traditional Japanese medicine,” “traditional medicine,” “ethnomedicine,” “folk medicine,” “folk remedies,” “indigenous medicine,” “primitive medicine,” “materia medica,” “homeopathic remedies,” “traditional East Asian medicine,” “traditional Far Eastern medicine,” “Far East medicine,” “Oriental medicine,” “Korean medicine,” “Tibetan medicine,” “herb,” “herbaceous agent,” “medicinal plant,” “medicinal herbs,” “medicinal plant product,” “plant preparation,” “herbal preparation,” “botanic,” “botany” “Kampo,” “traditional Mongolian medicine,” “Mongolian folk medicine,” “Mongolian medicine,” “phytotherapy,” “herb therapy,” “herbal therapy,” “ethnopharmacology,” “alternative medicine,” “alternative therapy,” “complementary therapy,” “complementary medicine,” “TCM,” “CHM,” “tang,” or “decoction”)  ] .P = 0.028; RR = 0.69, 95% CI = 0.55–0.88, P = 0.003), with no heterogeneity in grades III-IV  and I–IV   .P = 0.046; RR = 0.397, 95% CI = 0.242–0.650, P < 0.001; RR = 0.438, 95% CI = 0.294–0.654, P < 0.001; RR = 0.494, 95% CI = 0.291–0.839, P = 0.009), with no heterogeneity  analysis. The sensitivity analysis indicated that all analyses were robust with no evident publication bias.In this meta-analysis, the curative effect for grades III-IV was better than that for I–IV. The myelosuppression rate at toxic grades I-II showed no significant difference among the CHM treatment, no-CHM treatment, and placebo treatment groups (supplementary document). Result suggested that toxic grades I-II were responsible for the difference between toxic grades III-IV and toxic grades I–IV. We further analyzed the results for toxic grades I-II. In general, toxic grades III-IV were the index for the clinical treatment of patients showing myelosuppression, in whom treatment for toxic grade II or lower mainly focuses on symptomatic therapy. These results were similar to those in published articles about treating myelosuppression inpatients mostly at toxic grades III-IV. Source data from the 14 articles was insufficient to confirm our hypothesis about toxic grades I-II. We did not analyze toxic grades I, II, III, IV, and II–IV, as they provided no extra information.The main finding of our meta-analysis was different from that of the previous one , which iTo obtain robust results in the present meta-analysis, we included high-quality RCTs using Jadad scale to screen the methodological quality of articles and excluded studies that treated patients with acupuncture, intravenous CHM, CHM granules, patented CHM drug, or CHM extract, to reduce clinical heterogeneity among articles for comparison with previous meta-analyses. According to the theory for TCM, the herbs used in the 14 articles belong to yiqi-jiedu (tonifying qi and disintoxication) treatment. Our meta-analysis only included oral CHM; the results showed low heterogeneity  and homogeneity, which suggested that the yiqi-jiedu treatment could be the same treatment. To validate our hypothesis, we performed subgroup analysis of WBC at toxic grades III-IV . The 14 articles were divided by the herb into 4 subgroups, which means that yiqi-jiedu treatment was also subdivided into 4 treatments. The results were similar. Moreover, sensitivity analysis based on change of analysis model did not fundamentally alter most of the pooled results.In Asia, CHM has long been used in the treatment of chronic diseases and cancer as a primary therapy or adjuvant therapy. Nevertheless, one may assume that CHM played the role of placebo. Two of the 14 articles reported that the control group received placebo , 13, whiSome articles indicated that there was a positive interaction between the administered CHM and concomitant chemotherapy or radiotherapy , 29. TheNeutropenia is a common dose-limiting toxicity that is difficult to treat. Our meta-analysis indicated that oral CHM as an adjuvant can prevent the decrease in neutrophils during chemotherapy and radiotherapy, suggesting that CHM maintains the therapeutic dose and treatment cycle. Furthermore, CHM can prevent the decrease in WBC, HB, and PLT. Compared with the other treatments, the advantage of CHM is that it evidently improves the curative effect of chemotherapy and radiotherapy.Our meta-analysis has some limitations. First, CHM treatment is an individualized therapy. According to the theory for TCM, the difference in individualized therapy should be based on different diseases or symptoms. In our meta-analysis, the treatments in 14 articles belonged to yiqi-jiedu. Furthermore, our results show low heterogeneity and homogeneity and the results of subgroup analysis are similar. Although clinical heterogeneity is low, division into 4 subgroups could improve the robustness of the results. However, as the present research is limited, it was difficult to perform further analysis. Second, our meta-analysis did not include results for lymphocytes. Neutrophils: lymphocyte ratio is also an important hematological toxicity predictive index. However, there are no relevant articles meeting our inclusion criteria. Third, we included 5 types of cancers, with different drug sensitivity. Fourth, only two articles mention the concrete blind method. Fifth, we could not assess the impact of CHM treatment on other clinically meaningful endpoints, such as overall survival and quality of life, due to the limited number of reports available.Future studies could focus on the following points. First, drugs for tonifying qi and disintoxication play an important role in the treatment. Therefore, these types of drugs should be studied, as further subdividing treatment could provide more robust results, new therapy ideas, or drugs against myelosuppression. Second, better blind methods should be designed to improve methodological quality. Third, further studies can focus on other clinical endpoints, such as quality of life, survival, and length of stay, as there is a lack of high-quality studies demonstrating the correlation between CHM and these endpoints.Evidence obtained from this study suggests that CHM can be used as an adjuvant to alleviate myelosuppression induced by chemotherapy or radiotherapy and, in particular, reduce grade III-IV toxicity. However, due to heterogeneity and publication bias in the partial results of this meta-analysis , the results should be interpreted with caution and validated by conducting strictly designed multicenter RCTs of high quality and large scale.S1: PRISMA guidelines.


Borreliawas identified in Bothriocroton concolor and Ixodes holocyclus ticks from echidnas. Analyses of 16S rRNA and flaB genes identified three closely related genotypes of this bacterium (Borrelia sp. Aus A-C) that were unique and distinct from previously described borreliae. Phylogenetic analyses of flaB (763 bp), groEL (1537 bp), gyrB (1702 bp) and glpQ (874 bp) gene sequences and concatenated sequences (3585 bp) of three gene loci  were consistent with previous findings and confirm that this novel species of the genus Borrelia is more closely related to, yet distinct from, the Reptile-associated (REP) and Relapsing Fever (RF) groups. At the flaB locus, genotypes A, B and C shared the highest percentage sequence similarities  with B.orrelia turcica (REP), whereas at the groEL and gyrB loci, these genotypes were most similar (88.2–89.4 %) to B.orrelia hermsii (RF). At the glpQ locus, genotypes A and B were most similar (85.7 and 85.4 % respectively) to Borrelia sp. Tortoise14H1 (REP). The presence of the glpQ gene, which is absent in the Lyme Borreliosis group spirochaetes, further emphasises that the novel species of the genus Borrelia characterized in the present study does not belong to this group. Phylogenetic analyses at multiple loci produced consistent topographies revealing the monophyletic grouping of this bacterium, therefore providing strong support for its species status. We propose the name ‘CandidatusBorrelia tachyglossi’, and hypothesize that this species of the genus Borrelia may be endemic to Australia. The pathogenic potential of this bacterium is not yet known.Recently, a novel species of the genus  Spirochaetaceae is classified under the order Spirochaetales, belonging to the phylum Spirochaetes. This family consists of genera that are of concern to human health, such as Borrelia and Treponema [Treponema pallidum subsp. pallidum', the causative agent of syphilis worldwide [Treponema pallidum subsp. pertenue', the bacterium responsible for yaws [Borrelia is a member of the family Spirochaetaceae and through convention is divided into three major clades: Lyme disease/Borreliosis (LB) caused by members of the Borrelia burgdorferisensu lato complex, the Relapsing Fever (RF) borreliae and the Reptile-associated (REP) borreliae [Borrelia afzelii, Borrelia bavariensis, Borrelia burgdorferisensu stricto, and Borrelia garinii, along with a newly described genospecies ‘CandidatusBorrelia mayonii’ that causes LB in the upper Midwestern USA [Ixodes ricinus complex: Ixodes ricinus in Europe, Ixodes persulcatus in Europe and Asia, and Ixodes pacificus and Ixodes scapularis in USA [The family reponema , with coorldwide , and ‘Trfor yaws . The genorreliae . The LB tern USA . Memberss in USA . These ps in USA .Spirochaetes within the RF group have been reported throughout a number of continents, including Africa [Borrelia miyamotoi identified in I. persulcatus [I. ricinus [I. pacificus [I. scapularis [Borrelia lonestari‘' in Amblyomma americanum [Borrelia theileri in Rhipicephalus microplus [CandidatusBorrelia texasensis’ in Dermacentor variabilis [g Africa , Eurasiag Africa  and Nortg Africa . These bsulcatus , I. rici ricinus , I. paciacificus  and I. sapularis ; ‘Borrelericanum ; Borreliicroplus ; and ‘Cariabilis .Borrelia turcica in Hyalomma aegyptium ticks collected from tortoises in Turkey [Borrelia identified in various reptiles [The third major clade of this genus, the REP borreliae, was identified after the discovery of n Turkey , followereptiles . While tB. theileri and Borrelia anserina that are transmitted by Rhipicephalus (Boophilus) australis [Argas persicus [Borrelia queenslandica' was the first species of the genus Borrelia to be reported from native long-haired rats, Rattus villosissimus, in north-west Queensland, Australia. While the soft tick, Ornithodorus gurneyi, was considered to be the vector of this species due to its presence in the region, transmission experiments were not successful, and molecular characterization was never conducted to reliably identify the species of the genus Borrelia [Although LB borreliae have never been identified in Australian ticks, wildlife or people , two RF ustralis  and Argapersicus , respectBorrelia .Borreliaburgdorferi in 2008 [Sequence analysis of multiple loci offers the advantage over morphological characterization of being highly discriminatory, therefore serving as a reliable method for accurate identification, characterization and population, and epidemiological analyses in numerous bacterial studies . This te in 2008  and has  in 2008 .Borrelia was detected in a number of echidna ticks, Bothriocroton concolor, collected [Ixodes holocyclus tick [flaB, 400 bp) gene phylogenetic analyses, this species of the genus Borrelia formed a distinct clade from other well-described borreliae, indicating this organism, designated ‘Borrelia sp. Aus’, to be unique [flaB, groEL, glpQ and gyrB genes, in addition to the 16S rRNA and short flaB loci reported previously [Borrelia. Phylogenetic analysis confirmed its species status, and we hereby propose to designate this species as ‘CandidatusBorrelia tachyglossi’.Recently, a novel species of the genus ollected . An addilus tick . Based oe unique . In the eviously , to studAustralian Code for the Responsible Conduct of Research, 2007 and Australian Code for the Care and Use of Animals for Scientific Purposes, 2013. Tick collection was carried out opportunistically with the approval from the Murdoch University Animal Ethics Committee.This study was conducted under the compliance of the Bothriocrotonconcolor ticks, with 38 (39 %) ticks testing positive at the Borrelia-specific flaB locus [Borrelia-positive ticks were included in this study. Nested- and hemi-nested PCRs were conducted using primers targeting the housekeeping genes flaB (763 bp), groEL (1537 bp), glpQ (874 bp) and gyrB (1702 bp) . Primers were designed to amplify short fragments of each gene with overlapping regions in order to obtain maximum coverage of the genes analysed for accurate characterization. PCR cycling conditions were as described by Loh et al. [groEL primers: initial denaturation at 95 °C for 5 mins, 35 cycles of denaturation at 95 °C for 30 s, annealing at 48 °C for 40 s, extension at 72 °C for 2 mins, and a final extension at 72 °C for 7 mins. Amplification products of the targeted DNA products were electrophoresed in 1–2 % agarose gel with blue light using safe (Invitrogen), and positive samples were purified and sent for Sanger sequencing.Genomic DNA was extracted previously from 97 aB locus . These 3 groEL 15 bp, glpQflaB, groEL, glpQ and gyrB loci were aligned and analysed together with sequences representing species of the genus Borrelia retrieved from GenBank. All sequences were aligned using mafft v7.017 [muscle [mega6 [DNA sequences generated at t v7.017  and then [muscle . The bese [mega6  and was e [mega6 , and cone [mega6 . GTR andBorrelia-specific flaB (763 bp), groEL (1537 bp), gyrB (1702 bp) and glpQ (874 bp) DNA sequences were successfully amplified and sequenced from 38 Borrelia-positive Bothriocrotonconcolor ticks described by Loh et al. [flaB fragments; 24 samples were positive for all glpQ fragments; 13 samples were positive for all groEL fragments; and 10 samples were positive for all gyrB fragments.In the present study,  groEL 15 bp, gyrBBothriocrotonconcolor ticks using the 16S rRNA gene sequences, tentatively given the designations Borrelia sp. Aus A, Borrelia sp. Aus B and Borrelia sp. Aus C [CandidatusBorrelia tachyglossi’ genotypes A, B and C, respectively. At the flaB locus, genotypes A, B and C consisted of nine, three and five identical samples, respectively. The flaB gene alignment (787 bp) between ‘CandidatusBorrelia tachyglossi’ genotypes and other described species of the genus Borrelia showed that the novel genotypes shared highest percentage sequence identities with Borrelia turcica from the REP group (87.9–88 %); similarities to the LB Borrelia group ranged from 82.1–83.2 %; with the least similarity to Borrelia hispanica from the RF group (79.8–80 %). The percentage identities within the ‘CandidatusBorrelia tachyglossi’ genotypes ranged from 99.6–99.9 %. The percentage nucleotide identities between ‘CandidatusBorrelia tachyglossi’ genotypes and Borreliaturcica (87.9–88 %) were higher than that between Borreliaturcica and Borrelia hermsii (86.7 %). In contrast, the percentage nucleotide identities between ‘CandidatusBorrelia tachyglossi’ genotypes and Borreliahermsii (84.6–84.8 %) were lower than that between Borreliaturcica and Borreliahermsii (86.7 %) (Table S2). Phylogenetic analyses of the flaB gene locus showed that the ‘CandidatusBorrelia tachyglossi’ genotypes clustered most closely with Borreliaturcica with a high posterior probability  between ‘CandidatusBorrelia tachyglossi’ genotypes exhibited 83.9–85.7 % similarity with the REP species of the genus Borrelia and lower similarities (80.6–84.8 %) with the RF species of the genus Borrelia. The percentage similarity within ‘CandidatusBorrelia tachyglossi’ genotypes was 98.6 %. Phylogenetic analysis confirmed the closer relationship of the ‘CandidatusBorrelia tachyglossi’ genotypes from the present study with the REP Borrelia group (100 % bootstrap support) (CandidatusBorrelia tachyglossi’ genotypes and Borreliaturcica (84.1–84.2 %) and Borrelia coriaceae (RF) (84.1–84.8 %) were higher than that between Borreliaturcica and Borreliacoriaceae (83.3 %) (Table S3).At the support) . The pergroEL locus, ‘CandidatusBorrelia tachyglossi’ genotypes A and B were identical, and both shared 99.9 % similarities with ‘CandidatusBorrelia tachyglossi’ genotype C. Nucleotide alignment (1540 bp) between ‘CandidatusBorrelia tachyglossi’ genotypes and other described species of the genus Borrelia showed that the novel isolates had the least similarity with the LB group (83.9–85 %) and were most similar to Borrelia hermsii from the RF group (89.3–89.4 %). Phylogenetic analysis of the groEL locus showed that ‘CandidatusBorrelia tachyglossi’ genotypes from the present study clustered with the RF group with strong posterior probability support (CandidatusBorrelia tachyglossi’ genotypes and Borreliahermsii (89.3–89.4 %) were higher than that between Borreliahermsii and Borreliaburgdorferi (84.8 %); whereas the percentage identity between ‘CandidatusBorrelia tachyglossi’ genotypes and Borreliaburgdorferi (84.3 %) was slightly lower (Table S4). The genetic distance between ‘CandidatusBorrelia tachyglossi’ and Borreliaturcica at this locus remains to be determined until the groEL gene is characterized in Borreliaturcica.At the  support . The pergyrB locus, ‘CandidatusBorrelia tachyglossi’ genotypes A and B were identical. Nucleotide alignment (1712 bp) revealed that the ‘CandidatusBorrelia tachyglossi’ genotypes exhibited 80.8–82.0 %, 80.8–82.0 % and 84.8–88.2 % sequence similarity with the LB Borreliae, the RF group and Borreliaturcica, respectively. The percentage similarities within ‘CandidatusBorrelia tachyglossi’ genotypes ranged from 99.6 to 99.9 %. Phylogenetic analysis showed that ‘CandidatusBorrelia tachyglossi’ genotypes formed their own monophyletic clade, separate from Borreliaturcica and the RF group, with high bootstrap support (98 %) (CandidatusBorrelia tachyglossi’ genotypes and Borreliaturcica (87.8–88 %) and Borreliahermsii (88.1–88.2 %) were higher than that between Borreliaturcica and Borreliahermsii (86.7 %) (Table S5).At the t (98 %) . The perflaB and gyrB, available for each of the main borreliae  (Table S6). Likewise, the concatenated alignment (5154 bp), which excluded Borreliaturcica (REP), based on the four loci amplified in the present study  also produced a similar tree topology with ‘CandidatusBorrelia tachyglossi’ genotypes forming a monophyletic clade supported by high posterior probabilities , sharing the highest percentage identities with Borreliahermsii (90.3 %) (Table S7).A Bayesian phylogenetic tree reconstructed using the concatenated alignment (3585 bp) consisting of three genes, 16S rRNA, orreliae , illustrBorrelia as the closest sister clade. The recently established REP group has been detected in various reptiles from several countries and in ticks that parasitise them [Borrelia in Amblyomma varenense collected from the reticulated python (Python reticulatus) showed that the species of the genus Borrelia identified clustered together with the REP-associated Borrelia group, along with Borreliaturcica, based on phylogenetic analyses of 16S rRNA and flaB genes [Borrelia belonging to the REP group is unknown.All phylogenetic trees reconstructed revealed similar topologies with the REP group species of the genus ise them . The recaB genes . HoweverBorrelia are well known to cause diseases in humans outside of Australia; nonetheless, borrelial tick-borne disease in humans still remains highly speculative and controversial in this country. ‘CandidatusBorrelia tachyglossi’ was first reported in 2016 [Borrelia. Our results, based on sequence and phylogenetic characterization of multiple loci, provide conclusive evidence that ‘CandidatusBorrelia tachyglossi’, identified in Bothriocrotonconcolor ticks from echidnas, is distinct from other described species of the genus Borrelia and constitutes to a new clade in this genus.A number of members within the genus  in 2016 , hence tI. holocyclus, Bothriocrotonconcolor is a highly specialized tick, with echidnas as their primary host, and with a geographic distribution known only in Australia and Papua New Guinea [CandidatusBorrelia tachyglossi’ has previously been identified in one human-biting tick (I. holocyclus) removed from an echidna [I. holocyclus or other human-biting ticks remains to be determined. The morphological characteristics and the pathogenicity of this bacterium are also unknown.Borrelial spirochaetes are well known to be associated closely with wildlife and utilize tick vectors to maintain a sylvatic life cycle . Australw Guinea . ‘Candid echidna  and its CandidatusBorrelia tachyglossi′ .‘flaB, groEL, gyrB and glpQ). Comparisons of the flaB gene sequences among the ‘CandidatusBorrelia tachyglossi’ genotypes showed two single nucleotide polymorphisms (SNPs) in genotype A at bases 485 and 519 (GenBank accession no. KY586966); and one SNP in genotype B at base 227 (KY586964). As for the groEL gene, analysis revealed two SNPs in genotype C at bases 656 and 1143 (KY586970). Analysis of the gyrB gene showed two SNPs in genotype B at bases 985 and 1416 (KY586972); and five SNPs in genotype C at bases 405, 757, 1075, 1093 and 1420 (KY586973). At the glpQ locus, genotype A (KY586968) and genotype B (KY586967) showed 12 base differences at bases 3, 43, 103, 112, 264, 515, 520, 530, 532, 702, 819 and 852.Species can be differentiated from other borreliae based on sequence and phylogenetic analyses of five genomic loci (16S rRNA, flaB, groEL, gyrB and glpQ genes of ‘CandidatusBorrelia tachyglossi’ genotype A are 47.3, 40.9, 38.5, 33.8 and 34.1 mol%, respectively. The DA G+C contents for 16S rRNA, flaB, groEL, gyrB and glpQ genes of ‘CandidatusBorrelia tachyglossi’ genotype B are 47.2, 40.9, 38.5, 33.9 and 34.7 mol%, respectively. The DNA G+C contents for 16S rRNA, flaB, groEL, and gyrB genes of ‘CandidatusBorrelia tachyglossi’ genotype C are 47.2, 41, 38.5 and 34 mol%, respectively.The DNA G+C contents for 16S rRNA,


A number of studies had reported the association between tumor necrosis factor-alpha (TNF-α) gene polymorphisms and ischemic heart disease (IHD) risk. However, the results remained controversial. Therefore, we performed a systematic review with multiple meta-analyses to provide the more precise estimations of the relationship.We systematically searched electronic databases  for relevant studies published up to February 2017. The odds ratios (ORs) and 95% confidence intervals (CIs) were estimated for assessing the association. The present meta-analysis was performed using STATA 12.0 software.In total, 45 articles with 17,375 cases and 15,375 controls involved were included. Pooled ORs revealed a significant association between TNF-α −308G/A gene polymorphism and IHD  vs. GG: OR = 1.18, 95% CI = 1.03–1.36; (AA vs. (GA+GG): OR = 1.37, 95% CI = 1.08–1.75)), indicating that the TNF-α −308A allele might be an important risk factor for IHD. No association between other TNF-α gene polymorphisms and susceptibility to IHD were observed. No publication bias were found. Sensitivity analyses indicated that our results were stable.The present study indicated a possible association between the TNF-α −308G/A gene polymorphism and IHD risk. However, evidence was limited to confirm the role of TNF-α −238G/A, −857C/T, −863C/A, −1031T/C and other TNF-α gene polymorphisms in the risk of IHD. Generally, myocardial infarction (MI), unstable/stable angina (UA/SA), and coronary artery disease (CAD) were regarded as the main cardiovascular types of IHD. The major defining pathologic feature of IHD was atherosclerosis, and proinflammatory and inflammatory factors contributing to the process of atherosclerosis were thought to play an important role in the pathogenesis of IHD. Proinflammatory cytokine tumor necrosis factor-alpha (TNF-α), which was produced by inflammatory cells like monocytes, macrophages, and neutrophils, could stimulate cytokine secretion and augment the inflammatory response in turn. All these processes were involved in the formation, progression, and rupture of the atherosclerotic plaque, thus, changed expression of TNF-α might make great contributions to the process of IHD.Ischemic heart disease (IHD) was known as one of the major burden to the healthcare system for not only cost but also death and disability worldwide. Different polymorphisms of the TNF-α gene might cause different changes in the plasma level of TNF-α and take different effects in the course of IHD. To date, since the defect of the TNF-α gene was first studied in 1998, a large number of studies about the association between the TNF-α gene polymorphisms and IHD risk had been reported. Most studies were on the gene polymorphisms of TNF-α −238G/A (rs361525), −308G/A (rs1800629), −857C/T (rs1799724), −863C/A (rs1800630), and −1031T/C (rs1799964), while several studies were on the position of −376, −806, +476, +691, respectively.,9 Some studies demonstrated that the TNF-α gene polymorphisms could change the susceptibility to IHD.,11 However, other studies failed to confirm this relationship.,13The TNF-α gene was located on chromosome 6p21.3 in human and arranged within the class III region of the major histocompatibility complex (MHC).Therefore, in the present study, we performed a systematic review with multiple meta-analyses aiming to draw a reliable conclusion on the overall association between the TNF-α gene polymorphisms and susceptibility to IHD.2We used computer-based literature search strategy to identify potential studies that evaluated the association between TNF-α gene polymorphisms and IHD risk. The present study were reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement.2.1Electronic literature databases including PubMed, the Web of Science, EMBASE, Medline, Chinese National Knowledge Infrastructure, WanFang and ChongQing VIP Database were searched independently by 2 reviewers for relevant studies published up to February 2017 without restrictions on language or type of document. The following search terms were used: (“TNF” OR “tumor necrosis factor”) AND (“polymorphism” OR “genotype” OR “variant” OR “mutation”) AND . In addition, references of the selected publications or textbooks were searched manually as a source of relevant studies. The approval by an ethics committee is not required because the present study was based on published studies.2.2According to the inclusion criteria, 2 reviewers screened the relevant articles independently. Studies that met the following criteria were included in this meta-analysis: adult patients which were diagnosed with IHD; estimated the association between TNF-α gene polymorphisms and IHD risk; only cohort or case–control studies were included; had usable data on each genotype of both cases and controls. For reports on the same population or overlapping data, only the one with the largest sample size was included.2.3Information extraction of each eligible study was performed independently by 2 reviewers and disagreements were settled by a third reviewer. The following information were extracted from all eligible studies: the first author's name, publication year, study location, country, type of patient, ethnicity, source of control, genotyping method, study design, matching method between case group and control group, sample size together with percent of female, genotype distribution in both groups and Hardy–Weinberg equilibrium (HWE) evidence. the quality assessment of all included studies were performed by 2 reviewers independently. Any disagreement was resolved by a third reviewer. The scores of each study ranged between 1 and 9, and studies with the scores >6 were recognized as of high quality.In accordance with the Newcastle-Ottawa Quality Assessment Scale (NOS),2.4The genotype frequencies in controls of each study were tested by Chi-square test for HWE.The odds ratios (ORs) together with 95% confidence intervals (CIs) were used to estimate the association between TNF-α gene polymorphisms and susceptibility to IHD. Pooled ORs and 95% CIs were evaluated mainly according to 3 models which included allele model (B vs A), dominant model ((BB + AB) vs AA) and recessive model (BB vs (AB + AA)), respectively .I2 test was performed for evidence of heterogeneity among the results of different studies.I2 > 50% was considered as a sign of significant heterogeneity using the random-effects model, and I2 ≤ 50% was considered nonheterogeneity using the fixed-effects model. Subgroup analyses were conducted to find the potential sources for heterogeneity, based on ethnicity, quality, HWE, control source, matching method, sample size, and genotyping method. and Egger test was performed for the assessment of the potential publication bias. Sensitivity analysis was conducted by removing one study each time. P < .05 were regarded as statistically significant in all analyses. All statistical analyses were performed using the software STATA 12.0 .Begg test33.1A detailed description of the process of study selection is shown in Fig. 3.2The main characteristics of the included studies are shown in Table 3.33.3.1P = .005, I2 = 55.5%; (AA + GA) vs. GG: P = 0.002, I2 = 59.9%; AA vs (GA + GG): P = .555, I2 = 0.0%), random-effects models were used in allele model and dominant model, while fixed-effects model was used in the recessive model. The overall ORs demonstrated that there was no statistical association between TNF-α −238G/A gene polymorphism and IHD risk in neither genetic model  vs. GG: P < .001, I2 = 81.6%; AA vs (GA + GG): P < .001, I2 = 50.1%), thus the random-effects model were chosen in both 3 models. The pooled ORs revealed a significant association between TNF-α −308G/A gene polymorphism and IHD risk in allele model . Additionally, insufficient statistical power resulted from the much smaller sample size (only 5 studies with 1598 cases and 1499 controls were in Indians) could also help to interpret the distinct results from populations with different genetic backgrounds.,59Subgroup analysis according to ethnicity showed that, the TNF-α −308G/A gene polymorphism appeared to be associated with IHD risk in both Caucasians and Asians, but not in Indians. The most likely reasons lie in the population stratification within involved studies, especially when both allelic frequencies and incidence of disease vary across ethnic groups. There were 28 out of 44 studies using PCR-RFLP and 16 studies using other methods such as PCR-SSCP, MS, and TaqMan. Similarly to the total population, significant association between TNF-α −308G/A gene polymorphism and IHD risk was observed in the PCR-RFLP studies rather than the non-RFLP studies, suggesting that PCR-RFLP might be a good choice for genotyping method in DNA polymorphism analysis. Hence, when analyzing DNA polymorphism, we suggested using PCR-RFLP as a good choice of genotyping method according to the DNA sample size and the number of SNPs.We conducted subgroup analysis by genotyping method because of the increasing demand for precise diagnoses with advanced genotyping method. Therefore, the selection bias should be avoided in the case–control studies as far as possible. In addition, in a further subgroup analysis by sample size, a more significant association between TNF-α −308G/A gene polymorphism and IHD risk was observed in the group with the sample size <600 than in the group with the sample size ≥600. As described in other studies, small sample size studies were more likely to overestimate the effect of the genetic factors. Thus, future well-designed studies with larger samples are needed.In the subgroup analysis by control source, a significant association was observed among hospital-based studies only but not among population-based studies. This finding might due to the selection bias of the hospital-based case–control studies, because such controls might fail to represent the general population absolutely. (including 17 studies in 15 articles) and Wang et al (including 36 studies in 28 articles) only analyzing the association between TNF-α −308G/A gene polymorphism and IHD risk, our study had a more sufficient power to investigate the role of TNF-α gene polymorphisms in the risk of IHD, for 45 articles with 32,750 participants were involved. Except for the TNF-α −308G/A gene polymorphism, we also investigated the polymorphisms on the other position of TNF-α gene published up to date. What's more, we performed subgroup analysis by ethnicity, quality, HWE, control source, matching method, sample size, and genotyping method in the present study, which could help to identify the potential sources of heterogeneity.Compared with the meta-analyses conducted by Pereira et al According to our study, it is believable that TNF-α −308G/A gene polymorphism might be a suitable genetic marker for IHD risk. However, its presence under special environment circumstances  might be a key risk factor for IHD susceptibility. As described, the TNF-α genes were located within the region of highly polymorphic variation MHC and they were in linkage disequilibrium with each other and other genes, making the genetic influences caused by genes in linkage disequilibrium could not be avoided. Hence, additional method such as haplotypic analysis with other genetic markers may provide more useful data for the study of genetic etiology of IHD than current available single genotype-based data.Finding a certain link between inflammatory genetic markers and IHD requires a number of well-designed studies of phenotypically homogeneous subjects as well as multiple analyses of gene–gene and gene–environment interactions.Although some credible findings had been achieved, some limitations of our meta-analysis should be noted here. Firstly, the number of some included studies and the total sample size of some polymorphisms like −1031T/C polymorphism were relatively small, which restricted the statistical power for calculating a more accurate estimate about the association between TNF-α gene polymorphisms and IHD risk. Secondly, the ORs of all included studies were on the basis of unadjusted estimate, as not each included study reported an adjusted OR. Lastly, there was a lack of relevant studies about the association in Africans, Indians, and other ethnic groups, which restricted the power for finding the potential differences in the different ethnicities.In conclusion, despite the limitations, the present systematic review and meta-analysis indicated a possible association between the TNF-α −308G/A gene polymorphism and IHD risk, demonstrating that the −308A allele might be a risk factor for IHD. However, evidence was limited to confirm the role of TNF-α −238G/A, −857C/T, −863C/A, −1031T/C and other TNF-α gene polymorphisms in the risk of IHD. In order to confirm the current conclusion, future well-designed studies with larger samples are needed.We highly appreciated the support from all the participants.


Simultaneous multiple linear regression was used to predict 5 km time, using these determinants, indicating neither GXTHOT (R2 = 0.72) nor GXTCOOL (R2 = 0.86) predicted performance in the heat as strongly has previously been reported in cool conditions. vV̇O2max was the strongest individual predictor of performance, both when assessed in GXTHOT (r = −0.83) and GXTCOOL (r = −0.90). The GXTs revealed the following correlations for individual predictors in GXTHOT; V̇O2maxr = −0.7, RE r = 0.36, LT r = −0.77, LTP r = −0.78 and in GXTCOOL; V̇O2maxr = −0.67, RE r = 0.62, LT r = −0.79, LTP r = −0.8. These data indicate (i) GXTHOT does not predict 5 km running performance in the heat as strongly as a GXTCOOL, (ii) as in cool conditions, vV̇O2max may best predict running performance in the heat.In cool conditions, physiologic markers accurately predict endurance performance, but it is unclear whether thermal strain and perceived thermal strain modify the strength of these relationships. This study examined the relationships between traditional determinants of endurance performance and time to complete a 5-km time trial in the heat. Seventeen club runners completed graded exercise tests (GXT) in hot  and cool conditions  to determine maximal oxygen uptake (V̇O Under normothermic conditions, this behavioral response is highly associated with physiologic markers of endurance performance, such as lactate threshold (LT), lactate turnpoint (LTP), running economy (RE), and maximum oxygen uptake  that can be sustained. Running velocity is then determined by how efficiently the corresponding oxidative adenosine triphosphate turnover at the fractional utilization of V̇O2max is converted to locomotion . During an outdoor 16 km race, McLaughlin et al.2max, (vV̇O2max) explained 94.4% of the variation in performance time, while V̇O2max and LT each accounted for ∼90%.The determinants of the endurance performance model2max also appears to be important in determining absolute exercise intensity , with Périard & Racinais2max) in the heat, as in cool conditions. Therefore, individual determinants of endurance performance, such as the LTP and V̇O2max, appear to remain important for predicting endurance performance in the heat. However, specific thermal factors such as perceived thermal discomfort have also been proposed as determinants of self-paced exercise in the heat.SKIN) elicits concomitant effects on both perceived strain )Endurance running in hot and humid environmental conditions is characterized by elevated thermoregulatory, cardiovascular, and perceived exertional strain, which typically leads to a performance decrement, relative to cooler conditions.2max, LT, LTP, RE, vV̇O2max, and 5-km treadmill time trial (TT) performance, when the determinants are measured in both hot  and cool  conditions. The ultimate goal of this work is to inform on the appropriateness of using the traditional determinants of endurance performance model for research on endurance running in the heat.Therein, a heightened perceived strain may reduce the variation attributable to the traditional physiologic determinants, such that the specific determinants of endurance performance in the heat are not clearly defined. Such information would facilitate a more informed application of thermal interventions, such as precooling and heat acclimation, for alleviating physiologic versus perceived strain, but also aid performance prediction and training prioritization. Therefore, this study investigated the relationship between V̇OThis study on predicting 5-km running performance from the determinants of endurance performance is part of a recently published larger study of heat acclimation and performance.2max 61.0 ± 6.2 mL.kg−1.min−1, recent 5 km time 20:25 ± 1:42 min). Testing occurred in the UK spring, therefore participants were not naturally heat acclimated and were entering the competition season. The female participant completed all trials during the follicular phase of the menstrual cycle, verified via a questionnaire. Each participant provided written-informed consent and institutional ethical approval was issued in accordance with the Declaration of Helsinki (2013). Participants avoided intense exercise, alcohol and caffeine for 48 hours before testing and arrived hydrated, verified through urine analysis using a refractometer  in accordance with Sawka et al.Seventeen, amateur, club runners , who trained a minimum of three times per week for the previous 2 months, volunteered for this study , 27.2°C wet bulb globe temperature (WBGT)). During the second familiarization visit, participants completed a 5-km treadmill TT under the same environmental conditions. Experimental trials began 2 weeks after familiarizations to control against thermal adaptations from repeated visitsParticipants visited the laboratory on five occasions, comprising two familiarization visits and three experimental trials. On the first visit, a four-site skin fold caliper assessment was completed across the iliac crest, subscapular, triceps, and biceps, for the estimation of body fat percentage in accordance with the calculation of Siri.COOL; 13°C, 50% RH, 9.3°C WBGT) to assess the determinants of endurance performance . At least 2 d later, participants completed GXTHOT, the same test in hot and humid conditions . The final trial occurred 3–4 d later, where participants completed a 5-km treadmill TT in the same hot conditions. All trials were conducted in the same order and within an environmental chamber .During the first experimental trial, participants completed a GXT in cool conditions (GXT−1) within 6–8 exercise stages. Each stage involved 3 min of exercise, followed by 1-min rest, for blood sampling. Stages were separated by increments of 1 km.h−1. All tests occurred on a motorized treadmill  set at 1% incline, to replicate outdoor running.−1 below the previous final speed, with gradient increasing by 1% each min. Participants were not permitted to drink and were blinded to all forms of feedback.A graded exercise test, split into two parts, GXT 1 and GXT 2, was adopted, as described previously by Jones.ad libitum (increment 0.2 km.h−1) and total distance continuously displayed. The treadmill gradient was fixed at 1%. Participants were given no other feedback and did not drink during trials. Pilot testing within our laboratory, using a similar cohort, indicated a typical error for this trial of 16 s (1.2%).Upon arrival, participants rested in the hot environment for 10 min, before completing a 5 min, self-selected warm-up. Standardized instructions were given at the start of the trial and nothing thereafter, with participants instructed to “give your all,” “pace yourself throughout the trial,” and “adjust speed as you see fit” as per similar research.CORE) was measured using disposable rectal probes , inserted to 10 cm beyond the anal sphincter and connected to a data logger . Telemetry thermistors  were attached to the pectoralis major, biceps brachii, rectus femoris, and gastrocnemius for measuring TSKIN to calculate a weighted mean skin temperature.CORE, TSKIN, rating of perceived exertion . Core temperature  and lactate turnpoint (LTP), respectively.−1. Fingertip blood samples at the end of each stage were analyzed immediately . Ventilatory gases were measured using 30 s fixed-time averages , with the two values from the final min of each stage used for RE. Average RE (mL O2.kg−1.km−1) was calculated across the first five exercise stages, using the two fixed 30 s averages from the final minute of each stage. During GXT 2, the highest 30 s moving average represented V̇O2max. A V̇O2max, not V̇O2peak, was accepted when a V̇O2 plateau (< 2 mL.kg−1.min−1 across two successive 30 s fixed-time averages was observed).2max,2max decrement.−1, HR within 10 beats of age-predicted maximum, respiratory exchange ratio >1.1, and RPE at or above 19, as we have previously adopted.2max was calculated by multiplying V̇O2max (mL.kg−1.min−1) by 60 and dividing by the average RE.During the GXTs, running speeds at 2 and 4 mmol.lp<0.05 and presented as means ( ± SD). Initially, paired Student's t-tests were used to indicate differences in the number of V̇O2max and V̇O2peak tests, between conditions. Pearson's correlations (r) were used to examine for statistically significant relationships between the individual determinants of endurance performance and 5-km time. Following determination of a significant linear relationship, statistically significant variables were entered into a simultaneous multiple linear regression, with LT, LTP, RE, V̇O2max and vV̇O2max as the predictor variables and 5-km time as the dependent variable. The magnitude of difference between related, single samples is demonstrated by Cohen's avd, in accordance with the recommendations of Lakens.large, 0.5–0.3 moderate and 0.3–0.1 small, in accordance with Cohen.All outcome variables were assessed for normality and sphericity before further analysis. Data were analyzed using SPSS  with statistical significance set at 2max and RE . Consequently, correlations are derived from n = 17 for LT & LTP, n = 16 for V̇O2max & RE, and n = 15 for vV̇O2max. All predictor variables from both GXTHOT, GXTCOOL, and 5-km TT performance were normally distributed. Based on the predefined criteria, no difference (p = 0.08) was observed in the prevalence of V̇O2max and V̇O2peak tests between GXTCOOL  and GXTHOT .Technical faults resulted in no data for one measure of both V̇OHOT elicited a holistically elevated physiologic strain compared with GXTCOOL, as shown in HOT (−1 reduction in the LT and −0.7 (0.7) km.h−1 the LTP. V̇O2max was also impaired in GXTHOT (−4.6 ± 3.3 mL.kg−1.min−1), as was vV̇O2max (−0.6 ± 0.7 km.h−1). RE improved in GXTHOT, with the mean metabolic cost of running reduced by 12.3 ± 10.1 mL.kg−1.km−1, in the heat. Individual data demonstrating the impairments arising from heat stress to each of the determinants of 5-km performance are shown in Incremental running in GXTn in HOT , resultiPhysiological and perceptual responses from the 5-km TT in hot conditions are shown in HOT, LT, LTP, V̇O2max, and vV̇O2max were identified as statistically significant predictors of TT performance, but RE was not. The strongest individual predictor from GXTHOT was vV̇O2max (r = −0.83). These data are shown in  = 6.508, p = 0.008, R2 = 0.72, standard error of the estimate (SEE) = 106 s). The model revealed the following formula for predicting 5-km performance based on measures derived from GXTHOT.When measured in GXTCOOL, LT, LTP, RE, V̇O2max, and vV̇O2max were all statistically significant predictors of TT performance in the heat (2max was the strongest predictor of 5-km performance in GXTCOOL (r = −0.90). The results from GXTHOT and GXTCOOL are shown in HOT, that did not include RE in the predictive model, a simultaneous multiple linear regression was completed, excluding RE, revealing a significant relationship with TT performance . Subsequently, adding RE to these variables further improved the model . The model revealed the following formula for predicting 5-km performance based on measures derived from GXTCOOL.When measured in GXTthe heat . Again, HOT, the determinants explained 72% of performance and 82% in GXTCOOL. These findings indicate that the incremental running test in hot conditions is a weaker predictor of performance in the heat compared with testing in cool conditions. Supporting previous research conducted in cool conditions,2max appears the strongest predictor of 5-km performance in the heat. The overall models derived from both GXTHOT and GXTCOOL represent weaker relationships than are previously reported when the GXT and TT occur under conditions without heat stress,This study investigated the relationship between the determinants of endurance performance, when measured in hot and cool conditions, and 5-km running TT performance in hot conditions. In GXTHOT, vV̇O2max, V̇O2max, LT, and LTP explained 72% of the variance in 5-km performance, indicating that the majority of endurance performance in the heat is still underpinned by the traditional physiologic determinants. However, the prediction model reveals greater unexplained variance than previously reported for 16-km running performance (R2 = 0.9788) and did not include RE due to the absence of a significant linear relationship for RE to predict 5-km performance. It should be acknowledged there is likely a greater anaerobic contribution during a 5-km TT rather than 16 km,In GXTCOOL , indicating TT performance in the heat is not as closely associated to the physiologic responses during GXTHOT, as when the determinants are assessed in GXTCOOL. One explanation could be the fixed GXT intensities not eliciting analogous decrements on the determinants across participants, as demonstrated in HOT, relative to GXTCOOL, weakening the prediction model of GXTHOT. The differences between GXTHOT and GXTCOOL cannot be explained by the addition of RE to the model, because while RE strengthens the prediction of performance, the smaller model derived from V̇O2max, vV̇O2max, LT, and LTP already explained a greater proportion of variability and demonstrated a lower SEE in GXTCOOL. There were very high-sample correlations among many of these physiologic variables, in particular the running velocity variables, as has previously been reported when multiple independent variables are derived from the same individual.HOT and GXTCOOL, respectively.A stronger model was observed from GXT2max or RE, but appear slightly weaker than previous research in normothermic conditions , power output at 1 mmol.l−1, and power output at 4 mmol.l−1 respectively, to predict cycling performance in hot conditions . Based upon these data, Lorenzo et al.2max in elite athletesSKIN and TCORE than our participants experienced while running during GXT 1.Correlation coefficients between 5-km performance and blood lactate indices LT and LTP are stronger than for V̇Onditions . Notably2max and TT performance within runners of heterogeneous performance levels (r = −0.81–0.91),2max considered a pre-requisite for elite endurance performance, to meet the estimated energy requirements necessary to sustain running velocities observed in high-level competition.2max and performance may be weaker within an elite population due to the relative homogeneity of V̇O2max within this cohort,2max may still explain up to 59% of the variation in performance among elite marathon runners. The relationships between V̇O2max and performance in the heat  are weaker than that for 16-km running in the cool (r = −0.91),2max and running performance , unlike in GXTCOOL (r = 0.62), indicating RE assessment in hot conditions to be inappropriate for predicting performance in the heat. The influence of heat stress on RE is equivocal, possibly due to the progressive onset of heat strain and associated thermoregulatory responses, which may contribute to different outcomes between studies adopting different methodologies. For example, the relatively low starting intensities during the GXT may initially afford a benefit to RE through a more efficient muscle,HOT may not replicate the physiologic strain experienced during the TT, where a high intensity is observed from the outset (∼94% maximum HR), eliciting a faster and heightened thermoregulatory response, despite matched heat stress. It is also plausible that GXTCOOL elicited a small reduction in starting muscle temperature, affording a small impairment to muscle efficiency and requiring greater oxygen consumption for a given running speed, although we acknowledge muscle temperature was not measured. Notwithstanding, GXTCOOL demonstrated a stronger relationship between RE and performance, but this remains a relatively weak relationship, reaffirming RE to be a weak predictor of 5 km or similar distances.39 and would appear to exert the greatest influence within a group of athletes with relatively homogenous V̇O2max,2max range 51–75 mL.kg−1min−1).Running economy during GXT2max remained the strongest predictor, both when derived from GXTHOT and GXTCOOL, which is in broad agreement with previous literature in 2max to explain 92% of the variance in 8-km performance. This reaffirms the importance of training and monitoring of the parameters that determine vV̇O2max for improving 5-km performance in the heat. However, this observation should not necessarily reduce the importance attributed to blood lactate thresholds, given that Morgan et al.2max and determinants of blood lactate indices, such as capillary density, fiber-type distribution, respiratory capacity, and muscle enzyme activity, suggesting that training of either V̇O2max, RE, or lactate thresholds may provide mutual benefits.Despite the limitations of the relationship between RE and endurance performance, vV̇OHOT and GXTCOOL explained less of the variance in performance. When exercising under heat stress, it has previously been shown that perceived thermal and/or exertional strain exert marked influences on self-selected exercise intensity, in advance of changes in body temperature, through to moderate levels of hyperthermia.In comparison to the data of McLaughlin et al.,SKIN, with TCORE exerting a greater influence as hyperthermia progresses.CORE elevation and hyperthermia became more pronounced, physiologic strain may have influenced performance both directly, through limiting aerobic capacity, but also indirectly by enhancing RPE.Behavioral thermoregulation has previously been suggested to be a determinant of exercise performance under heat stress.As a 5-km TT was not completed in cool conditions, the efficacy of determinants of endurance performance for predicating performance for this cohort outside of heat stress is unknown. It is also acknowledged that exercise tests were conducted without representative air flow, which will likely impair convective cooling2max, measured in cool conditions. The traditional determinants of endurance performance, vV̇O2max, V̇O2max, LT, and LTP, appear to remain prerequisites, accounting for 82% of variance in performance when measured in GXTCOOL, emphasizing the importance of continuing to train these areas. However, our study also alludes to prioritizing improved perceived thermal and/or exertional strain, to minimize behavioral attempts to thermoregulate. Therefore, monitoring thermal sensation, comfort and RPE relative to fixed velocities and durations during an athlete's training program in a hot environment  would appear useful to track improvement. Furthermore, both short- and long-term acclimation training appear effective strategies for improving perceived thermal strain,Event characteristics such as distance and duration may determine whether it is appropriate to conduct a laboratory test in representative environmental conditions, due to the potential for heat stress to afford a transient improvement to running economy that does not appear to replicate 5-km TT exercise. Therefore, completing TT in the heat may be more appropriate for assessing training status in competitive athletes before competing. When laboratory testing does take place, the best single predictor of 5-km performance would appear to be vV̇OCOOL appears more appropriate than GXTHOT, possibly due to the progressive onset of heat strain not replicating that of the time trial. The vV̇O2max also appears to remain the best predictor, when running endurance performance occurs in the heat. Finally, the model of the traditional determinants of endurance running performance; vV̇O2max, V̇O2max, RE, LT, and LTP, appear pre-requisites for endurance performance in the heat, but may explain less variance in performance than previously reported in cool conditions, albeit over longer distances.In conclusion, predicting running performance in the heat from GXT


Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and β-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.We have prepared mouse fur extensively The online version of this article (doi:10.1007/s10858-016-0056-7) contains supplementary material, which is available to authorized users. Keratinous tissues, including skin, hair, fur, nails, claws, hooves, and feathers, play essential and varied biological roles as connective, structural, protective, and insulating materials. They are important as sources of raw materials, in medical and veterinary therapy, as well as in cosmetic industry and science. Relationships between keratin chemical composition Robbins , molecul13C NMR suggests that the former is ca. 40 % α-helical, the latter random coil  has been applied to characterize, compare, and tentatively assign spectra from hair, hoof, and feathers. The secondary structure content is predicted to be highly heterogeneous, with α-helical rod domains co-existing with β-sheet, random coil, and turn, structures in the keratin head and tail domains and in the matrix proteins. This heterogeneity is reflected in the partial resolution of the amide carbon signal into higher frequency (α-helix) and lower frequency (β-sheet/random coil) components  are low natural abundance, selective, or total, isotopic enrichment is necessary. This is usually achieved by producing target biomolecules in microorganisms which will overexpress the desired construct on isotopically enriched growth media. Unfortunately, isotope enrichment of mammalian biomaterials is not yet possible this way. However, we have recently produced highly and generally enriched murine tissue and biomaterials in reasonable time and at acceptable cost  supplemented with standard laboratory animal diet . At the time of weaning of the pups, all animals are culled and tissue harvested. This procedure produces high levels of protein isotope enrichment, and is particularly advantageous for labelling slower turnover tissues such as fur, quite adequate for 2D NMR. In bone, these experiments produce data which reflect chemical composition and protein structural diversity in unprecedented detail  was obtained. However at 9.4 T there is insufficient spectral dispersion to aid structural analysis.CP-MAS 13C–13C correlation (DQ—SQ) experiment via POST-C7  axis of the chemical shifts of the two bonded atoms. Chemically equivalent, or near equivalent, atoms that happen to be mutually bonded thus give rise to a cross peak in the DQ dimension at twice the chemical shift of each equivalent atom. In this way, for instance, the chemically very similar and mutually bonded Phe ring carbons give rise to the broad signal envelope centred at ca. 130 ppm , and produce an “autocorrelation” peak at the corresponding DQ coordinate of ca. 260 ppm. In contrast, the more spectrally dispersed Tyr ring carbons give rise to resolved sets of cross peaks corresponding to ε−ζ , δ−ε  and 130 ppm (δ) in SQ dimension), while the cross peak between Tyr γ and δ overlaps the cross peaks from Phe δ, ϵ and ζ, at ca. 260 ppm (DQ) and 130 ppm (SQ). The expected cross peak between the quaternary Phe γ (the high frequency shoulder at ca. 137 ppm) and δ is most likely too broad and weak to be observed, as is the case for the expected Phe and Tyr γ−β connectivities. The much less abundant His and Trp residues are not observed with any certainty.Figure Partial splitting of the amide signals into two components (ca. 172.5 and 175 ppm in our samples) is ascribed to the co-existence of β-sheet/random coil, and α-helical, secondary structures respectively  mixing time maps out single bond aromatic ring proximities, while a longer (100 ms) mixing time reveals longer range interatomic crosspeaks such as Cδ−Cζ, and ring carbon–amide carbon. The effects of mixing time are exemplified in Fig. S6.2D PDSD experiments  results in correlation “signals” which are well resolved in one or both of the 2D experiments, and which potentially report on the chemical environment of each atom in the amino acid chain. The effective increase in resolution in the DQ dimension of the SQ–DQ correlation experiment is particularly useful, and resolves other residues apart from those already alluded to: Pro, Ile, Leu, and Val.Resolving the spin systems of certain amino acids allows some inferences regarding their secondary structural environment. Taking advantage of the fact that the principal secondary structure families  exert opposite effects on Cα, and Cβ, chemical shifts, the difference between them , subsequently re-forming by oxidation (peroxides), and disrupting non-covalent networks and hydration by treatment with alkali, acid, or heat. Potentially any of these effects on molecular structure can be interrogated by NMR, with resolution greatly enhanced by the types of labelling approach described here. The resolution of the structurally important Tyr residues is particularly enhanced by the non-specific labelling we have described, assuring that changes in Tyr environment should be reported by NMR. Unfortunately in our universally labelled material signals from other structure-critical residues  overlap with too many other signals to be immediately useful markers of structural or chemical changes. However it is possible to modify the feeding protocol to limit the incorporation to specific labelled amino acids, or at least to these amino acids and their metabolic products in the case of the non-essentials.In conclusion, general isotope labelling of keratin materials enables the application of powerful 2D NMR techniques which offer significant progress in determining the molecular structures and intermolecular interactions underlying its material properties. Furthermore the 2D approaches provide resolution enhancement which enables signals of a number of specific residue types to be observed and resolved. In the future this will enable a deeper understanding of the structural basis of industrial and cosmetic processes, and assist biomimetic design of new artificial materials.Supplementary material 1 (DOCX 2720 kb)Below is the link to the electronic supplementary material.


Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the “RAP tag” system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification. In contrast, protein tags sometimes affect the character of the target proteins if they are large in size (>25 kDa); therefore, their removal is necessary before any protein analysis. Peptide tags are less likely to affect the structure and function of target proteins because of their small size ; therefore, it is not always necessary to remove the tag portion for protein analysis.A TARGET tag, PA tag, and MAP tag, among many others. These systems sometimes have disadvantages, such as low specificity, low affinity, or difficulty in achieving protein elution. Therefore, we need to develop further affinity tag systems to overcome these disadvantages of established affinity tag systems.The most appropriate peptide tag system can be selected from many available peptide tags, such as FLAG tag, Podoplanin is a type I transmembrane protein, which is highly expressed in many normal cells and cancer cells, and is involved in tumor-induced platelet aggregation by binding to CLEC-2 on platelets.,25–33 Because PMab-2 possesses high affinity and specificity against rat podoplanin, it was expected to be useful as an antitag antibody. Herein, we developed a novel affinity tag system, the “RAP tag” system, using PMab-2 mAb.We previously developed a mouse mAb (clone PMab-2) against the platelet aggregation-stimulating domain of rat podoplanin.ak = 2.0 × 105 M−1 s−1, dk = 2.0 × 10−3 s−1, and KD = 9.7 × 10−9 M, indicating that PMab-2 showed moderate affinity toward the RAP tag. Next, we examined whether the RAP tag system is useful for several protein detection systems. As depicted in  PMab-2 did not show any nonspecific bands in CHO-K1 and LN229 cells, indicating that PMab-2 is very specific to the RAP tag. PMab-2 reacted with the EGFR with the N-terminal RAP tag, which was expressed in CHO-K1 cells in flow cytometry (ec) with RAP tag was added at a concentration from 3 ng/mL to 10 μg/mL and detected by biotinylated NZ-1. As shown in ec was detected in a dose-dependent manner. These results indicate that the RAP tag system is useful for Western blotting, flow cytometry, and sandwich ELISA.We first investigated the binding affinity between PMab-2 mAb and RAP tag (DMVNPGLEDRIE) using the BIAcore X100 system. Curve fitting showed the affinity for PMab-2–RAP tag interaction: ytometry , left. Aec and the soluble ectodomain fragment of human epidermal growth factor receptor 2 (HER2ec) were expressed in LN229 and were purified from the culture supernatant  or 2–3 (HER2ec) at a high concentration (ec and HER2ec are highly glycosylated,35; therefore, they were electrophoresed at a “heavier” position compared with that suggested by the theoretical molecular weight. ATRXepi was eluted in elution fractions 2–10 (Next, we purified three different RAP-tagged proteins using the RAP tag system. The EGFRernatant . The fral lysate . All RAPntration . EGFRec ons 2–10 . These pIn conclusion, we successfully developed a novel affinity tagging system, “RAP tag,” by employing a unique mAb PMab-2 against rat podoplanin. The RAP tag system could be advantageous for protein purification and detection in the field of protein science.