Osteoporosis, a common chronic metabolic bone disease is associated with considerable morbidity and mortality. As the prevalence of osteoporosis increases with age, a paralleled elevation in the rate of incident fragility fractures will be observed. This narrative review explores the origins of bone and considers physiological mechanisms involved in bone homeostasis relevant to management and treatment. Secondary causes of osteoporosis, as well as osteosarcopenia are discussed followed by an overview of the commonly used pharmacological treatments for osteoporosis in older people.The online version contains supplementary material available at 10.1007/s40520-021-01817-y. Osteoporosis, a disease characterised by low bone mass and microarchitectural deterioration of bone tissue is the most common chronic metabolic bone disease. Annually contributing to 8.9 million fractures worldwide as well as to reduced physical and psychological health, lower quality of life and shorter life expectancy, osteoporosis represents a major global health problem –3. In thTo understand the mechanism by which osteoporosis develops and the treatment options available, an understanding of the development, structure and remodelling process of bone in addition to the effects of ageing, disease and drug treatments on bone is needed. In this narrative review, our aim is to explore bone physiology and homeostasis, pathology and diagnosis of primary and secondary osteoporosis, osteosarcopenia and management of osteoporosis relevant to clinical practice.During gastrulation, the blastula differentiates into three distinct cell lineages: the ectoderm, mesoderm and endoderm. At week four, the mesenchymal cells of the intraembryonic mesoderm divide into the three paired regions: paraxial mesoderm, intermediate mesoderm, and the lateral plate mesoderm . The latBone is a specialised and multifunctional connective tissue with both an organic and inorganic component. The organic bone matrix (osteoid) is comprised of collagenous proteins, the predominant being type I collagen  as well Of the two main types of bone in the adult skeleton, cortical comprises approximately 80% of adult bone mass and trabecular, the remaining 20%. Cortical bone is dense, has a low turnover rate of around 3% per year and maintains mechanical strength and integrity of the bone , 8. In cThe cellular component of bone is chiefly composed of three cells: osteocytes, osteoblasts, and osteoclasts. Osteocytes are found in the lacunae of the matrix and have a mechano-sensory function in bone formation. Osteoblasts synthesize osteoid whilst osteoclasts enzymatically resorb bone . All thrBone remodelling allows repair of micro-damage, maintaining skeletal structure as well as serum calcium and phosphate homeostasis and involves a careful equilibrium between the action of osteoclasts, tightly coupled with that of osteoblasts. Numerous clusters of these cells within multicellular units are found along the bone surface forming active remodelling sites which are individually covered by a cell canopy. This cell canopy has been found to be derived from mesenchymal stem cells (MSC) surrounding the red bone marrow and acts as a source of progenitor cells during the remodelling process . A remodActivation of the resting bone surface is mediated by osteocytes that express an amino acid peptide; receptor activator of nuclear factor (NF) Kappa-B ligand (known commonly as RANKL), which interacts with the RANK receptor on osteoclast precursors that potently induces differentiation into multinucleated osteoclasts. Osteoblast expression of macrophage colony-stimulating factor (M-CSF) also promotes osteoclast precursor survival and differentiation. Osteoblasts produce chemokines, to recruit osteoclast precursors, and matrix metalloproteinases to degrade un-mineralised osteoid and expose adhesion sites for osteoclast attachment –13.Osteoclasts secrete hydrogen ions and lysosomal enzymes, e.g., cathepsin-K into a ‘sealed zone’ beneath the cell , 15. ThrThe reversal phase has been subject to intense research in recent years , 17 and Osteoblasts deposit un-mineralised osteoid until the tunnel of resorbed bone is completely replaced, resulting in minimal net change in bone volume during remodelling . Bone foWhen the tunnel of resorbed bone has been fully replaced, the remodelling cycle ends through a series of yet undetermined termination signals. The resting bone surface is re-established.Remodelling signals may be hormonal or mechanical in nature. Systemic regulators of bone formation include oestrogen, growth hormone and androgens. Thyroid hormones are essential for normal musculoskeletal development, maturation, metabolism, structure and strength where they promote bone turnover by influencing osteoblast and osteoclast activity . GlucocoBone volume and mass decline in older individuals and in all ethnicities. An imbalance in remodelling within the aged microenvironment is driven by MSC senescence and a shift in differentiation potential to favour adipogenesis within the bone marrow. An altered intracellular signalling milieu such as lower Sirtuin levels can lead to an increase in sclerostin activity, with inhibition of wnt and suppression of bone formation. Increased activity of mTOR translates to increased osteoclastic activity and release of cathepsin K .2D3), in the kidneys by 1a-hydroxylase (CYP27B1). PTH stimulates 1α-hydroxylase to increase levels of calcitriol. When serum calcium levels are normal or low, calcitriol acts on vitamin D receptors (VDRs) to increase intestinal and renal calcium uptake. However, when dietary calcium is insufficient to meet calcium demand, i.e., during periods of undernutrition often seen in older people, a negative calcium balance ensues. At this juncture, calcitriol inhibits bone mineralisation and enhances bone resorption through upregulation of RANKL expression. Through these actions, calcium and phosphate are mobilised from bone matrix to serum, at the expense of skeletal integrity. Calcitriol activation of osteocyte VDRs results in increased production of fibroblast growth factor 23 (FGF-23) which inhibits 1α-hydroxylase, thus creating a negative feedback system ) receptors. 5-HT may have important signalling and regulatory roles in bone remodelling. SSRI use in women with a mean age of 78.5 years was associated with an increased rate of non-spine fractures . In oldeHyperthyroidism can lead to higher bone turnover and osteoporosis. Both endogenous (primary thyroid disease) and exogenous causes, i.e., long term therapeutic use of levothyroxine as well as over-replacement with levothyroxine is associated with lower BMD and a subsequent increased risk of hip and vertebral fractures , 34, 47.LD are commonly prescribed and used in older people. Their diuretic activity centres around sodium and chloride reabsorption at the loop of Henle but they also decrease calcium reabsorption and increase calcium excretion. Hypocalcaemia can lead to increased bone turnover and lower BMD . In clinThe incidence of CKD increases in older age, but also in those who are hypertensive and have diabetes. CKD is associated with osteoporosis and renal osteodystrophy through perturbations in 1α-hydroxylation of vitamin D in the kidney, hypocalcaemia and hyperparathyroidism. Detailed discussion on renal bone disease is out of scope for this review. Several recent articles offer detailed reviews .Both type 1 (T1DM) and type 2 (T2DM) diabetes are risk factors for low-energy fractures as both types are associated with low bone quality and strength, though only T1DM typically reduces BMD. T1DM is associated with increased risk of fracture throughout the lifecourse particularly at the hip , even whT2DM is not associated with decreased BMD but is still associated with increased fracture risk at the hip, vertebrae and other vulnerable sites . This prGiven bone and muscle share similar embryonic origins, both tissues may be influenced by similar metabolic (endocrine and paracrine) and environmental cues to maintain homeostasis. Sarcopenia (muscle failure) is characterised by a decline in skeletal muscle strength, mass and function . PrimaryThe pathophysiology of osteosarcopenia is multifactorial; the common mesenchymal origins of bone and muscle infer a close relationship in their pathogenesis . As suchThe diagnosis of osteoporosis is made using DXA scanning to measure the bone mineral density (BMD) of the proximal femur to obtain a T-score. The T-score represents the number of standard deviations (SD) a patient’s BMD is below the mean reference value of a healthy young population. A T-score ≤ 2.5 SD below the reference value indicates osteoporosis  and wherThe UK National Institute for Health and Care Excellence (NICE) advise that all women aged 65 and above, all men aged 75 and above, and younger patients with risk factors should receive a form of osteoporosis risk assessment. The gold standard investigation for osteoporosis is bone mineral density (BMD). BMD at the femoral neck, age, sex, smoking, family history, and the use of oral glucocorticoids can be used to calculate the FRAX score. This tool estimates the 10-year probability of osteoporotic-related fracture . The TraScreening and intervention for individuals who are high risk of fracture as a primary preventative endeavour could reduce the burden of future fragility fracture. For example, screening with FRAX and pharmacological intervention for post-menopausal women aged 70–85 at high risk developing a fracture was associated with a decrease in hip fracture rate and was deemed to be cost effective compared with usual care in the UK SCOOP study . Older pRecently the concept of imminent fracture has been developed to highlight those most at risk of fracture within 2 years after a sentinel fracture. These events can occur in up to 23.2% of patients  and riskThe diagnosis of sarcopenia relies on case finding through administering the SARC-F questionnaire or determining the presence of weaker grip strength through hand-held dynamometry or poorer performance in repeated chair rises. A probable case of sarcopenia identified at this stage allows multicomponent intervention with either nutritional or physical activity interventions. Measurement of lean mass through DXA and other measures of physical function such as gait speed can determine whether an individual has severe sarcopenia .Research supports physical activity and exercise for the prevention of osteoporosis and related injury from falls and fractures , 75. In 1. Calcium and vitamin D2. Antiresorptive therapy—Bisphosphonates, Denosumab.3. Hormonal treatment—Selective oestrogen receptor modulators, Testosterone, PTH analogues.4. Novel therapies—Romosozumab, Dickkopf-1 (Dkk1) inhibitors.There are various pharmacological options for osteoporosis treatment that aim to reduce the risk of fractures. These include:Vitamin D deficiency in older people is common, not only secondary to physiological changes in the ability of the skin to synthesise vitamin D but particularly in those who are malnourished, have chronic kidney disease, are institutionalised or are housebound. National guidance recommends 1000 mg of calcium in combination with 400 International Units (IU) of vitamin D per day. Housebound older people or those living in a nursing home are advised to take 800 IU of vitamin D per day. A meta-analysis found that calcium and vitamin D supplementation reduced the risk of hip fracture by 30% and the total fracture risk by 15% . This waEvidence opposing the use of calcium supplementation suggests an increased risk of cardiovascular disease, including myocardial infarction . HoweverBisphosphonates bind strongly to hydroxyapatite, inhibit osteoclast-mediated bone resorption and increase bone mineral density. They are associated with beneficial effects on lowering the risk of fractures amongst a broad age range of patients; even those living with frailty –94. EvidUK NICE guidance  recommen2 and 30 mL/min/1.73 m2, respectively. However, eGFR may not be accurate in older people, especially those living with frailty and sarcopenia. Cockcroft and Gault estimation of GFR is, therefore, appropriate to use in these situations; especially when IV Zoledronic is being considered . Atypical femoral fractures can occur particularly after 5 years of bisphosphonate use at the rate of 1:1000/year. Oral bisphosphonates should be taken on an empty stomach, in an upright position, with a glass of water . Adheren2. FREEDOM , a large multicentre placebo-control trial showed a reduction in fracture incidence of 68% for vertebral fractures, 40% for hip fractures, and 20% for non-vertebral fractures, in the first 3 years, in postmenopausal woman taking Denosumab [Denosumab is a humanized monoclonal antibody that blocks RANKL and hence osteoclastic activity. It is given via a subcutaneous injection (60 mg)on a 6-monthly basis alongside calcium and vitamin D supplementation in individuals with a GFR > 30 ml/min/1.73 menosumab ; 10 yearenosumab . DenosumWhen initiating Denosumab or other anti-resorptive therapy, it is important to ensure that patients have normal serum calcium levels and are replete in vitamin D . This loSelective oestrogen receptor modulators (SERMs) such as Raloxifene and Lasoxifene aim to prevent bone resorption due to oestrogen deficiency. They are indicated primarily for the treatment and prevention of osteoporosis in post-menopausal women and are indicated after first line therapies have been considered. As an example of efficacy, Lasoxifene 0.5 mg showed 42% reduction in vertebral fracture risk and 24% reduction in hazard rates of non-vertebral fractures, at 3 years in women aged 59–80 years , 87. MosEndocrine Society recommends testosterone for men at high risk for fracture with testosterone levels below 200 ng/dl (6.9 nmol/l). This should be considered even for patients who lack standard indications for testosterone therapy but who have contraindications to other osteoporosis’ therapies . PotentiTeriparatide, a synthetic parathyroid hormone, is anabolic in bone rather than anti-resorptive. It can be used in men and women who are intolerant or who suffer severe side effects from first line therapies described. Teriparatide should be administered subcutaneously, 20mcg daily for a maximum of 24 months. Teriparatide is contraindicated in patients with metabolic bone diseases such as Paget’s disease, skeletal muscle metastases or previous bone radiation therapy. Side effects include nausea, pain in limbs, headache and dizziness. Abaloparatide, a newer PTH analogue showed lower risks of new vertebral fractures when compared to both placebo and teriparatide as well as lower risk of nonvertebral fractures in comparison to placebo and a significant increase in BMD amongst 2463 post-menopausal women aged 49–86 years in the The ACTIVE study   and 27% lower risk of clinical fractures (p < 0.001). The risk of nonvertebral fractures was lower by 19% (p = 0.04) and the risk of hip fracture was lower by 38% (p = 0.02). Nonetheless, it is important to note an imbalance in serious cardiovascular adverse events between the 2 groups—16 patients (0.8%) in the Romosozumab group vs 6 (0.3%) in the alendronate group reported cardiac ischemic events ; and 16 patients (0.8%) in the Romosozumab group vs 7 (0.3%) in the alendronate group reported cerebrovascular events . Further studies are needed to clarify this imbalance [The ARCH study, however, compared a group of postmenopausal women that received alendronate for 24 months and a group that received Romosozumab for 12 months followed by alendronate for another 12 months. Interestingly, patients on the Romosozumab-to-alendronate group had a 48% lower risk of new vertebral fractures Below is the link to the electronic supplementary material.


Nitrous acid (HONO)is an important component of the global nitrogencycle and can regulate the atmospheric oxidative capacity. Soil isan important source of HONO. [HONO]*, the equilibrium gas-phase concentrationover the aqueous solution of nitrous acid in the soil, has been suggestedas a key parameter for quantifying soil fluxes of HONO. However, [HONO]*has not yet been well-validated and quantified. Here, we present amethod to retrieve [HONO]* by conducting controlled dynamic chamberexperiments with soil samples applied with different HONO concentrationsat the chamber inlet. We show a bi-directional soil–atmosphereexchange of HONO and confirm the existence of [HONO]* over soil: when[HONO]* is higher than the atmospheric HONO concentration, HONO willbe released from soil; otherwise, HONO will be deposited. We demonstratethat [HONO]* is a soil characteristic, which is independent of HONOconcentrations in the chamber but varies with different soil watercontents. We illustrate the robustness of using [HONO]* for quantifyingsoil fluxes of HONO, whereas the laboratory-determined chamber HONOfluxes can largely deviate from those in the real world for the samesoil sample. This work advances the understanding of the soil–atmosphereexchange of HONO and the evaluation of its impact on the atmosphericoxidizing capacity. EquilibriumHONO concentration over soil, [HONO]*, existsas a soil characteristic, which is a key parameter for quantifyingsoil−atmosphere HONO fluxes. Under atmosphericallyrelevant conditions, the uptake coefficient of NO2 (γ)on aerosols such as mineral dust,23 soot,20 and organic particulates21 is at magnitudes of <10–6, while γof >10–4 to 10–5 is requiredtoexplain the observed HONO formation rates of 0.2–2.0 ppb h–1.27 Additional HONO formation mechanismsuch as the photosensitized reduction of NO2 on humic acidsurfaces has been proposed.28 Moreover, laboratorystudies found that heterogeneous HNO3 photolysis on aerosolsexhibited a high HONO production rate and has been accounted as animportant HONO source.31 However, multiple scattering effects of light onaerosol sample filters used in those experiments may lead to an overestimationof the observed reaction rates.33The main source of atmospheric HONO has been a mystery fordecades.Emission from combustion processes1 After production from nitrification and denitrification, soil nitriteactively participates in the reversible acid–base reaction[NO2– (aq) + H+ (aq) ⇔HNO2 (aq)] and releases HONO to the atmosphere throughliquid–gas partitioning [HNO2 (aq) ⇔ HONO(g)]. Recently, several more studies have been conducted examiningsoil HONO fluxes42 and the laboratory chamber fluxes were directly used as estimatesof fluxes in the real world.42 One problem of this treatment is the different transfer/depositionvelocities of HONO in the laboratory and real-world conditions, whichwill lead to different fluxes under these two conditions. Up to now,there has been a lack of knowledge on how to translate measured fluxesin the laboratory chamber to those in the real world. In addition,most laboratory measurements have focused on measuring only HONO emissionfrom soil by applying HONO-free air at the chamber inlet.40 However, HONO deposition to soil should also be taken into accountas it can occur at high atmospheric HONO concentrations. Micrometeorologicalfield measurement methods of HONO fluxes, such as eddy correlation(EC), have been developed to directly determine HONO fluxes in thefield.45 However, it is stillproblematic due to the lack of rapid and sensitive techniques to measureHONO fluxes.44 Empirical parameterizationand process-based modeling is a labor-efficient alternative and severalmodels have been used to simulate soil HONO fluxes.50 Empirical parameterization models have calculated HONO emissionsbased on laboratory-determined chamber HONO fluxes as a function ofsoil water content (SWC).51 A different approach has been suggested by Su et al.1 based on the resistance model,54 as shown in 2 (aq)] in the soil, [HONO]atm is the atmospheric HONOconcentration, and vt represents the transfer/depositionvelocity of HONO. The resistance model approach, being analogous toelectrical current resistance,54 accountsfor three major processes that limit the transport of HONO from/tosoil surfaces including (i) turbulent transport between the atmosphereand the top of the so-called quasi-laminar layer, a very thin layerof stagnant air adjacent to the soil surface, (ii) molecular transportacross the quasi-laminar layer, and (iii) emission or deposition from/tothe soil surface. Accordingly, three resistances in series, that is,the aerodynamic resistance Ra, the quasi-laminarlayer resistance Rb, and the surface resistance Rc govern the HONO transport .55vt equals to the reciprocal of the total resistance . A major difference between 1 If [HONO]*, as a soilcharacteristic, indeed exists, we argue that Besides chemicalreactions, Su et al. showed that biogenic soilnitrite can be an important HONO source.In this study, we aim to demonstratethe existence of [HONO]* oversoil, develop a method to derive [HONO]* by controlled dynamic chambermeasurements, and quantify the atmospheric soil HONO fluxes. Moreover,[HONO]* variabilities during soil drying processes are investigated.Table S1.Soil samples were collected on 03 Aug 2020from an agricultural wheat field in Mainz, Germany (49°59′33.7″N8°13′05.5″E), at a depth of 0–5 cm. Thecollected samples were air-dried, grinded, and sieved through a 2mm cutoff stainless-steel sieve and stored in the dark at room temperaturefor 3 months before analysis. The physicochemical properties of thesoil sample are shown in –1, corresponding to 31% water holding capacity (WHC)of the soil. The sample was then placed into a dynamic flow-throughchamber. The chamber had an inner diameter of 12.0 cm and a heightof 13.0 cm. The inner wall material of the chamber was a 50 μmthin transparent Teflon film (FEP) . To control the temperature of the soil sample,the inner volume of the chamber bottom plate (made of PVDF) was continuouslyflushed with water cycled using a thermostat . The chamber was purged with purified air derived bypassing ambient air through an ozone generator to oxidize nitrogen-containingtrace gases, followed by sequential filter columns filled with glasswool , silica gel ,Purafil , and activated charcoal . Theinlet purging air was humidified using a PID-controlled split (dry/wet)gas system comprising two mass flow controllers  and RH sensors . Downstreamof the humidification step, HONO gas was added to the inlet purgingair at a small flow rate of 0.02–0.04 mL min–1 controlled using another mass flow controller. Many methods havebeen used to generate stable HONO.56 Here,HONO gas was generated by flushing purified air through the headspaceof a HONO source solution, which was prepared by dissolving NaNO2 (1.25 mM) in a citric acid buffer (pH = 4) solution. Thechange in the HONO source concentration was less than 0.1 ppb within∼10 h . From a totalairflow of 6.9 L min–1, only a fraction of 2.6 Lmin–1 was used to purge the chamber, as this amountwas consumed using three gas analyzers. In the overflow exhaust pipeupstream of the chamber, a needle valve was installed. This variableflow resistance was used to keep the inner chamber volume at a slightlyhigher pressure than ambient, to prevent the risk of laboratory aircontaminating the chamber. A Teflon-coated fan was installed in thecenter of the chamber lid to sustain highly turbulent conditions withinthe chamber.The soil sample wasprepared in a Petri glass dish containing 50 g of soil and 25 g of pure water (18.2 MΩ). Thesample was thoroughly mixed by mechanical stirring and placed in drypurified air at room temperature to reach an SWC of 0.12 kg kg57 The gas-phase H2O concentration was measuredusing an infrared CO2/H2O analyzer operatedin the differential mode . The variation of SWC was calculated using the measured differentialwater vapor concentrations between the chamber inlet and outlet ata given time (DLicor) and the differenceof the mass of the soil  prior to and after  the HONO exchange experimentHONO was measured with a commercial long path absorptionphotometer . Theestimated uncertainty of HONO measured by LOPAP is ∼10%. Thelower detection limit was calculated from two times the standard deviationof the zero air signal (2σ) at ∼40 ppt for 1 min averages.The LOPAP technique is explained in detail elsewhere.t = 0 denotesthe time when the measurementstarted, t = N is the time whenthe soil dried out, and t = n isany time between t = 0 and N. msoil,d is the mass of the oven-dried soil, whichwas determined by putting the soil sample in an oven at 110 °Cfor 24 h after the HONO exchange experiment. The wall loss of H2O and HONO was corrected according to a reference measurementwhen the chamber was empty. The flow chart of the chamber system isshown in Figure S3.Here, Q, and exits the chamberat the outlet. The HONO flux (F) of soil is relatedto the transfer/deposition velocity (vt) of HONO and the gradient between the HONO concentration of thechamber bulk headspace air (Ccham) andthe equilibrium HONO concentration over the soil surface, [HONO]*,1 here defined as C*In the dynamic flow-through chamber,continuous purging air enters the chamber at the inlet, purges thechamber at a flow rate of To be noted, under ideal conditions,when the equilibrium in the soil is reached, [HONO]* over the soilsurface would be the same as that in the soil. In practice, the equilibriumis expected to be reached within a shallow topsoil layer.F) can also be quantified usingthe chamber mass balance equation58TheHONO flux . Therefore, Here, Ccham) can be assumeduniform as the chamber air was well-mixed and hence also equals tothe concentration measured at the chamber outlet (Cout)To be noted, Combining Cin1 of HONO concentrationat the chamber inlet and measuring the concentration at the chamberoutlet (Cout1), the following equationis obtainedWhen applying Cin2, the concentration at the chamber outlet(Cout2) was measured. A similar equationas When HONO concentration atthe chamber inlet was switched to Cout2 werethe same as during the determination of Cout1 , and thus,parameters on the right-hand side of Here, soil conditions during A and vt) are canceled out and C*can be solved from eq In this way, theunknown parameters . The RH* results showedno dependence on different chamber turbulent conditions  and the mean of the RH*results of all tests was 97.4%. The consistency of mean RH* underdifferent chamber turbulent conditions and the proximity of the RH*to 100% confirm the validity of the C* method, thatis, retrieving C* from a set of two different inletconcentrations while monitoring the respective outlet concentrations.The method is applicable to obtain equilibrium concentrationsnotonly of HONO but also of other trace gases over soil and other surfaces.Because the equilibrium relative humidity (RH*) of air over a liquidwater surface is known to be 100%, a chamber test with liquid waterin a Petri dish was performed to validate the applicability of theabovedescribed –1 (10% WHC), the HONO concentrations decreased. This pattern of soilHONO emission during the soil drying process is similar to that foundin previous studies.36To see the influenceof inlet HONO concentrations on the soil HONOfluxes, the outlet HONO concentration data were evaluated independentlyfrom each other according to the three inlet HONO concentrations applied2. At an 1 Up to now, only theoretical [HONO]* values havebeen calculated according to pH and nitrite content of bulk soils.59 In this work, we introduced a method to retrieve the actual [HONO]*values during the soil drying process (see the *1) and grouping 5 and 15 ppb ([HONO]*2). As shown in 1 and [HONO]*2. Theseresults show that the retrieved [HONO]* is independent of the inletHONO concentrations applied, which proves that [HONO]* indeed existsas a soil characteristic. As shown in out), HONO will be released from the soil; otherwise, HONOwill be deposited to the soil. In the real world, the comparison between[HONO]* and atmospheric HONO concentrations can predict whether HONOis emitted from or deposited to the soil. Furthermore, [HONO]* isstrongly dependent on SWC as shown in –1 (10% WHC) and then [HONO]* decreased as SWC further decreased. WhenSWC decreases, the increasing concentration of HNO2 (aq)in soil water can lead to a higher [HONO]*, according to Henry’slaw behavior of gas–liquid partitioning [HNO2 (aq)⇔ HONO (g)]. This, however, could not explain the decreasein [HONO]* when SWC further decreases. A possible explanation is thelimited kinetic mass transport and the nonideal solution behaviorat lower SWC.60[HONO]*, the equilibrium gas-phase HONO concentrationover thesoil, has been suggested to be an important parameter to determinethe bi-directional HONO exchange between soil and the atmosphere.–2 s–1 at different SWCs during thesoil drying process . Chamber H2O vapor fluxes were observed to be strongly dependent on chamberturbulent conditions. On the other hand, as noted above in the [HONO]*method section, the turbulent conditions did not significantly affectthe observation-based RH* ,which indirectly demonstrates the robustness of the [HONO]* method.The chamber HONOflux from/to the soil sample was calculated according to  process 5A. In prvt) and the atmospheric concentration of HONO([HONO]atm) are also crucial parameters for quantifyingsoil HONO fluxes according to vt depends primarily on themeteorological conditions and soil resistances, and the reported vt values fall in the range of 0.077–3cm s–1.65 Field observations of [HONO]atm have been extensivelyperformed (Table S3) and up to ∼10ppb of [HONO]atm have been reported for a fertilized agriculturalfield site in the North China Plain.66 [HONO]*at different SWCs during the drying process of an agricultural soilsample was determined by the present study  to 538.7 ng m–2 s–1 (when[HONO]atm = 0 ppb), as shown in Figure S8. These results show that the predicted atmospheric HONOfluxes can differ widely from HONO fluxes measured in the chamber, [H2O]out is the measured H2O concentration at the chamber outletwith inlet dry air, and [HONO]out is the measured HONOconcentration at the chamber outlet at an inlet HONO concentrationof 0 ppb. In Figure S9, [HONO]* derivedby the [H2O] method shows a good agreement (within 30%)with that derived by the [HONO]* method of the present study. Thissuggests that the transfer/deposition velocities, vt, of HONO and H2O vapor are similar in thesame chamber system. vt equals to thereciprocal of the sum of Ra, Rb, and Rc . Ra, the aerodynamicresistance, is the same for surface–air exchange of these twospecies in the same chamber system. Smaller [HONO]* values derivedby the [H2O] method than those by the [HONO]* method couldbe explained by the smaller Rb, quasi-laminarlayer resistance, and Rc, surface resistancefor the water–air exchange of H2O vapor than thosefor soil–air exchange of HONO. For the [HONO]* method, twosets of HONO measurement results under the same soil and chamber conditionswere used for [HONO]* calculation, which guaranteed that Ra, Rb, and Rc were all the same. [HONO]* can then be solved accurately.Meusel et al. estimated[HONO]* by a simplified method using measurementsof the water–air exchange of H1 Assuming an SWC of 0.04 kg kg–1, the theoretical [HONO]* of the soil sample at theexperiment temperature (22 °C) was calculated to be ∼0.3ppb according to its nitrite content (0.43 mg kg–1) and pH (7.7) measured before the soil drying process. In comparison,the observation-based [HONO]* at an SWC of 0.04 kg kg–1 was ∼30.8 ppb . Both[HONO]* and [HONO]atm can be affected by various environmentalfactors. For example, high temperature during daytime leads to increased[HONO]* due to the temperature dependence of the equilibrium of HNO2 dissociation and gas–liquid partitioning on the soilsurface.1 At the same time, the photolysisof HONO under sunlight causes a relatively low daytime [HONO]atm. As a result, [HONO]* is larger than [HONO]atm during daytime and HONO emission from the soil should prevail. Inthis scenario, soil will be a daytime HONO source, which helps usto explain the missing HONO sources observed during daytime.68 During nighttime, a decreased [HONO]* at low temperature and a relativelyhigh [HONO]atm due to the absence of photolysis can leadto HONO deposition to the soil and soil will be a HONO sink. Therefore,such diurnal variations of temperature and sunlight could lead todiurnal patterns of HONO fluxes between soil and the atmosphere. Ourpredicted pattern is in accordance with the observed diurnal HONOfluxes in an agricultural field.66 Besidestemperature, other parameters can also influence [HONO]*, such asSWC, soil nitrite, pH, and microbial activity. Although there is anincreasing body of field measurements of [HONO]atm, experimentalinvestigations and model simulations on [HONO]* are still requiredto unravel and quantify soil HONO fluxes under different environmentalconditions. In addition, [HONO]* is also linked with soil moistureand chemical, physical, and biological processes in the soil. We recommendfurther studies to investigate the dependence of [HONO]* on HONO gas–liquid/gas–solidexchanges and the kinetic mass transport in the soil. It is also feasibleto apply the [HONO]* method in the field to derive [HONO]* of soilwith its original properties and thickness. Investigations of [HONO]*could improve our predictions of atmospheric HONO fluxes of soil andour understanding of how the biosphere affects air quality and globalclimate.The present study shows thatthe exchange of HONO between soil and the atmosphere is bi-directionaland provides direct evidence of the [HONO]* existence over soil. Thissoil–atmosphere exchange of HONO is mainly regulated by [HONO]*and the atmospheric HONO concentration ([HONO]


Kalidium schrenkianum is a perennial halophyte growing in an arid habitat with radiation stress (137Cs) in northwest China. In this study, K. schrenkianum growing under different environmental stresses were selected to investigate the dynamics and persistency of endophytic microbial communities amid seasons in a year. The results showed that Gammaproteobacteria and unassigned Actinobacteria were the most dominant bacterial communities, while the most dominant fungal communities were Dothideomycetes, unassigned Fungi, and Sodariomycetes. The bacterial community diversity in roots was higher than that in aerial tissues, and root communities had higher diversity in summer and autumn. In contrast, the fungal community diversity was higher in aerial tissues comparing to roots, and the highest diversity was in spring. Season was a determinant factor in the microbial community composition in the roots but not in the aerial tissues. RaupCrick index suggested that the bacterial communities were mainly shaped by stochastic processes. Our research investigated the community traits and members with temporal persistency. For example, bacterial taxa Afipia, Delftia, Stenotrophomonas, Xanthomonadaceae_B_OTU_211, and fungal taxa Neocamarosporium F_OTU_388, F_OTU_404, F_OTU_445, and unassigned Fungi F_OTU_704, F_OTU_767 showed higher frequencies than predicted in all the four seasons tested with neutral community model. The networks of co-occurrence associations presented in two or more seasons were visualized which suggested potential time-continuous core modules in most communities. In addition, the community dynamics and persistency also showed different patterns by radiation levels. Our findings would enhance our understanding of the microbial community assembly under environmental stress, and be promising to improve the development of integrated concept of core microbiome in future.Endophytes are essential components of plant microbiota. Studies have shown that environmental factors and seasonal alternation can change the microbial community composition of plants. However, most studies have mainly emphasized the transitive endophyte communities and seasonal alternation but paid less attention to their persistence through multiple seasons.  Endophytes are microbes living within healthy plant tissues with no adverse effects on plant health . They arIonizing radiation comprises high-energy electromagnetic waves or particles that can ionize an atom or a molecule. These ionizing radiations exist in the natural environment and are a form of abiotic stress to organisms. Environmental radiation alters the physiology and morphology of the organisms in radioactive environments and causes negative effects on the ecosystem . NeverthThe core microbiome concept is commonly used in microbiome studies to accommodate community members or features playing important roles in community structure, assembly, maintenance, or function. Although its definition varies due to specific backgrounds and purposes in different studies, the term “core” is committed to depicting the shared features of microbial communities . For exa137Cs) accumulation in the arid, saline, and semi-arid desert of northwest China. Seasonal sampling was conducted to collect the aerial tissues and roots of Kalidium schrenkianum  Moq. , which is a dominant population of local flora, to investigate the effect of seasonal changes and radiation levels on endophytic microbial communities (bacteria and fungi). This study aimed to reveal: (1) the changes in endophytic communities in response to seasonal alternation; (2) the influence of environmental radiation stress on seasonal dynamics of endophytic communities; (3) the shared features of endophytic communities across the four seasons.Endophytes colonize host plants and jointly challenge harsh environmental conditions, including radiation stress . However137Cs accumulation, attributed to natural precipitation and concentration within alluvium of seasonal floods. The other site is 8 km away and not affected by radionuclide (control site). Moreover, the soil had a salt content above 2% on the surface layer (<20 cm deep). Halophyte K. schrenkianum was the most dominant plant population in the local habitat, especially at the radionuclide contaminated site.Sample collection was conducted in Hoxud County in the Xinjiang Uyghur Autonomous Region of China . The area has a semi-arid climate with a mean annual temperature of 12.56°C and mean annual precipitation of 591 mm. Two sites located in a watershed area of seasonal floods were selected for sample collection. One site is a radiation-contaminated region with Kalidium schrenkianum plants were collected from two sites with different radioactivity levels in April (spring), July (summer), September (autumn), and December (winter) of 2018. Plants materials were collected using a random sampling approach from a 50 m × 50 m square plot in each site. Five plants per plot were randomly selected (at least 15 m apart) and uprooted. Whole plants were labeled and placed in large autoclaved paper envelopes, then transported to the laboratory in an icebox. The samples were then stored at −80°C until further use. Additionally, five soil samples were collected in September 2018 (autumn) from the surface soil (0–20 cm deep) along the diagonal of the square plot using shovels. A total of 40 samples (2 radiation levels × 4 seasons × 2 tissue types × 5 plant replicates) were used for endophyte analysis. The soil samples from each site were sieved to remove rocks and plant litter, thoroughly mixed, labeled, and packed in cloth bags, and stored at 4°C to transport to the laboratory.137Cs in soil samples was analyzed at the Northwest Institute of Nuclear Technology . One hundred grams of each soil sample were put into an environmental source box (ϕ 45 cm × 25 cm plastic box) and analyzed on an HPGe γ spectrometer . Each soil sample was placed 8 cm away from the detector, and the measurement time ranged from 1 to 3 days. The 661 keV peak of 137Cs in soil samples was measured and analyzed by net counting, and the radionuclide activity of 137Cs in the soil was calculated  was surface sterilized with 75% (v/v) ethanol for 1 min, 3.25% (w/v) sodium hypochlorite for 3 min, and 75% (v/v) ethanol for 30 s  ethanol and dried using SpeedVac2  for 10 min or until dry. The DNA pellet was resuspended in 100 μl TE buffer . The DNA concentration and quality were measured using a NanoDrop 1000 Spectrophotometer .Total DNA was extracted from the aerial parts and roots of for 30 s . Genomicfor 30 s . Briefly® High-Fidelity PCR Master Mix ; 0.2 μM of forward and reverse primers, and about 10 ng template DNA. Thermal cycling conditions were as follows: initial denaturation at 98°C for 1 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, then a final extension at 72°C for 5 min. The PCR products were mixed with an equal volume of 1× loading buffer (containing SYB green) and separated via 2% agarose gel electrophoresis. The expected bands were cut from the gel and purified using GeneJETTM Gel Extraction Kit . Sequencing libraries were generated using Ion Plus Fragment Library Kit 48 rxns , according to the manufacturer’s instructions. The library quality was assessed on the Qubit@ 2.0 Fluorometer . Finally, the library was sequenced on an Ion S5TM XL platform.The V5–V7 hypervariable region of the bacterial 16S ribosomal RNA gene was amplified using 799F (AACMGGATTAGATACCCKG)  and 1193NGS sequencing yielded 79 samples for bacterial dataset and 78 for fungal dataset, as one sample failed to acquire sufficient reads of bacteria and two samples failed for fungi. Raw reads from the bacterial and fungal dataset were demultiplexed and quality filtered using QIIME software (Version1.7.0). Reads with a quality score <20 and those lacking complete barcode and primers were excluded from further analysis. Chimeric sequences were removed using USEARCH software. Subsequently, DADA2 workflow  was usedThe bacterial and fungal taxa were determined using the pipeline described by MZ911967-MZ912492  and MZ919361-MZ919966 . Also, the raw sequences have been deposited in the Sequence Read Archive of NCBI under BioProject PRJNA757585  and PRJNA757584 .The ASV and OTU sequences have been deposited in the GenBank of National Center for Biotechnology Information under the accession numbers Reviewer link:PRJNA757585https://dataview.ncbi.nlm.nih.gov/object/PRJNA757585?reviewer=etp8sv9to4q0cie9dldjc40tgdPRJNA757584https://dataview.ncbi.nlm.nih.gov/object/PRJNA757584?reviewer=3o9ugraugup7lrg9io6chhgtr5Taxon agglomeration yielded a bacterial and fungal dataset of 2,234,413 and 3,272,864 reads, respectively. Subsequently, singletons and doubletons were filtered from the datasets, and taxa with <0.001 relative abundance were removed to reduce the noise. This resulted in a bacterial dataset of 565 taxa from 79 samples and a fungal dataset of 606 taxa from 78 samples. The bacterial and fungal datasets were then rarefied to 7,500 and 7,400 reads, respectively; the minimum value of read sums among all samples of the dataset. Finally, a bacterial dataset comprising 564 taxa and 79 samples and a fungal dataset containing 606 taxa and 78 samples were generated.metaMDS function in “vegan” package  using the “iNEXT” package  and Raup-Crick index (RCI) were used to evaluate the stochasticity in the assembly of communities . Bacterial model . Calculaal model . RCI wasal model .rho statistic was used to estimate correlation with function cor.test in “stats” package  presented within four seasons were recorded to reveal the association persistency among seasons. If a co-occurrence association between certain taxa presented in two or more seasons, it was kept in a combined network for visualization. The combined co-occurrence networks were visualized with “igraph” package  dominated the aerial fungal communities and showed dramatic colonization preference to sites  and roots . Season alternation significantly influenced bacterial community compositions in the roots , but not in the aerial tissues . ANOSIM results were similar to those obtained by PerMANOVA, reflecting the same patterns of environmental effects on bacterial community compositions. Radiation stress and season alternation did not affect aerial bacterial communities. However, seasonal changes affected bacterial communities in the roots . Furthermore, seasonal alternation had no significant effect on fungal communities in aerial tissues  or root . ANOSIM results showed that only the site had significant effect on the fungal community compositions in the aerial tissues  and Fungi spp. (F_OTU_704 and F_OTU_767) exhibited higher frequencies than predicted in all the four seasons, except in the roots at the radiation stressed site . Divergent distribution in structural similarities between networks was found, especially for fungal communities . These results are similar to those reported by previous studies. However, the highest stochasticity of fungal communities was found in summer. Regarding bacterial communities, only those in the roots showed increasing stochasticity from summer to winter. One possible reason is that annual plants assemble microbiota a fresh to begin their growth cycles, while the microbiota assembly of perennial K. schrenkianum is affected by the endophytes which have already colonized the plant. Therefore, such taxa exert their first-colonizer-priority and interfere with the community assembly of endophytes in the new growth cycle  can be found in the article/JZ, XS, and Z-DZ designed the experiments. JZ and Q-YT carried out the experiments. JZ and XS analyzed the experimental results. JZ, XS, and Z-DZ wrote and reviewed the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Scaffolds used to receive stem cells are a promising perspective of tissue regeneration research, and one of the most effective solutions to rebuild organs. In the near future will be possible to reconstruct a natural tooth using stems cells, but to avoid an immune-defensive response, sterilize the scaffold is not only desired, but also essential to be successful. A study confirmed stem cells extracted from rat’s natural teeth, and implanted into the alveolar bone, could differentiate themselves in dental cells, but the scaffold’s chemistry, geometry, density, morphology, adherence, biocompatibility and mechanical properties remained an issue. This study intended to produce a completely sterilized dental scaffold with preserved extracellular matrix. Fifty-one samples were collected, kept in formaldehyde, submitted to partial demineralization and decellularization processes and sterilized using four different methods: dry heating; autoclave; ethylene-oxide and gamma-radiation. They were characterized through optical images, micro-hardness, XRD, EDS, XRF, SEM, histology and sterility test. The results evidenced the four sterilization methods were fully effective with preservation of ECM molecular arrangements, variation on chemical composition (proportion of Ca/P) was compatible with Ca/P proportional variation between enamel and dentine regions. Gamma irradiation and ethylene oxide presents excellent results, but their viability are compromised by the costs and technology’s accessibility (requires very expensive equipment and/or consumables). Excepted gamma irradiation, all the sterilization methods more than sterilizing also reduced the remaining pulp. Autoclave presents easy equipment accessibility, lower cost consumables, higher reduction of remaining pulp and complete sterilization, reason why was considered the most promising technique. Considered the most mineralized tissue of the human body, enamel is composed basically of crystalline PAH (Hydroxyapatite—near 90%) in structure Ca10(PO4)6(OH)2, water and organic matter (protein and lipids). The hardness of the enamel is between 3.2 and 4.4 GPa or 270 to 360 VHN and in relation to the fatigue strength it is considered friable, usually presenting micro fractures by stresses beyond its elastic limit. Tensile strength is close to 10 MPa and compressive strength is approximately 262 MPa. Its modulus of elasticity in compression tests reaches 33.6 GPa4. Dentin is a biological composite of a collagen matrix filled with nanometric apatite crystallites deficient in Ca , rich in carbonate, F (iron) and Mg (magnesium), and scattered on parallel hollow cylinders called canaliculated or dentinal tubules3. The dentin has tensile strength is 50 MPa and hardness from 50 to 60 VHN 4.The tooth’s design is a marvel of engineering, as it is capable to absorb static and dynamic energies5. To determine the suitability of scaffolds, it is important to consider: biocompatibility, biodegradability, mechanical properties, scaffold architecture, and manufacturing technology. The recovery of organic functions, through the use of stem cells, constitute the most challenging task, when viable, the ideal substitute should be from autologous origin6.Tissue engineering is one of the most challenging areas in regenerative medicine, more than replacing damaged tissues, it is expected the restoration, maintenance or improvement of the regenerated tissue9. Although ceramic scaffolds exhibit excellent biocompatibility, due to similarity with hydroxyapatite (HA), the applications are limited by the brittleness and difficulty to sharp, in addition new bone formed in a porous HA cannot sustain the mechanical loading needed for remodeling or the compressive forces on chewing (near 700 N)10. These difficulties in terms of structure, functionality and mechanical properties, when producing scaffolds from single phase biomaterial, encouraged research with composite materials, as ceramic-polymer or metal-ceramic based scaffolds13.Scaffolds can be produced from biological, synthetic or composite materials, the natural materials are more biologically active, usually promote excellent cell adhesion, growth, and their biodegradability allows to host cells, consequently, stimulate the production of its’ own ECM (Extracellular Matrix). The use of synthetic materials as polymers and ceramics are growing significantly, the polymers can be tailored built and biodegradation can be controlled through the composition, although still exist the risk of infection, due to reduced bioactivity. It is growing the use of polymer or ceramic based prosthesis, for example knees or shoulders, metal-ceramic is also present on teeth’s restoration or dental crowns16. The bioscaffolds can be used in tissue regeneration with ability to carry out cellular activities, drugs and genes, produced with biocompatible material, absorbable or not. As the structure is very complex, being composed by solid, fiber and gel, the best scaffold would be the natural one, because it was already able to keep living cells in activity17. Gupte and Ma18 defend the materials should bio-mimic scaffolds’ features, developing characteristics for cell-bearing and create an environment to improve cell adhesion, proliferation, differentiation and also to promote the tissue regeneration. More than design, the mechanical integrity and functionality, morphology and surface compatibility are also essential for cell adhesion, differentiation, and integration with surrounding natural tissues. Additional care need to be taken with sterilization, to avoid infection and rejection21.The scaffolds, being tridimensional structures similar to original tissue in texture, macro and micro-geometry, make easier the cellular adhesion and consequent expression, the ECM can influence the cell’s behavior, and the mechanical properties are critical to mechanic transduction, development, differentiation and regeneration22; the drivability of human dental stem cell to immature osseous tissues, including tooth beak of chick embryo23 can be a promising technique. Another well succeeded experiment, using dental pulp stem cells seeded on collagen scaffolds (in vitro), were able to produce a fresh stemmed pulp tissue25.To produce a complete tooth from stem cell remains a challenging task, however, the production of collagen sponges and gels, used for tooth regeneration, has shown great compatibility to retain cells and support proliferation, differentiation, and also stimulate the formation of calcified tissues16, being partially successful on the production of a teeth. Although there was a visible separation of each layer  the shape remained an issue, incentivizing the study of sterilized teeth as a bioscaffold, what supposedly would improve the results. The total cells removal from a tissue or an organ (decellularization) is a complex process, as there are structural and functional proteins constituting the ECM, if it is damaged, can compromise the adequate expression of stem cells. If used in excess of time or concentration, the demineralization and decellularization solutions may interfere on ECM and alter its ultra-structure, on the other hand, if biological contents are not completely removed, can cause an inflammatory or immune response. The pulp removal could be done through an endodontic access (perforation), but this method was not used in order to keep the ECM preserved.The idea of producing a natural tooth from stem cell has been tried seeding stem cells on a rat5. Lack of vascularization in scaffolds and tissue engineered constructs is a major problem27, an alternative to vascularization is engineering microvasculature by stem cells previously to implantation29.Although good results are reached developing mineralized dentin, enamel and crown through the use of tissue engineering, the complex structure and interactions between the epithelium and mesenchyme tissues justify the difficulty to develop a viable organ from stem cells31. The host's reaction to a foreign body can be inflammatory response or an immediate and immune rejection, being one of the reasons the use a biomaterial scaffolds, obtained through decellularization of tissues and organs, should provide better results. The conservation of ECM are well tolerated by xenogeneic receptors, to use biomaterial scaffolds, but it is recommended an adequate sterilization process to be implanted32.Decellularized tissues are extensively used to obtain biomaterial scaffolds, where the efficiency of cells removal depends of the origin and the methods used to sterilize it. The ideal conditions to use these tissues include the minimum interference on biochemical composition, ultra-structure and the mechanical behavior of ECM. The elimination of microorganisms as bacteria, spores, protozoa, fungi, and virus are essential to reach an acceptable level of safety on sterilization17. Although exist several different methods, were chosen 4 methods well known36 to be compared in this study. The autoclave sterilization (AS) is the most used in dental clinics, although the thermo-sensitivity of several hospital materials do not allow the generic use of this method33. In the past it was common to use the dry heat (DH) method, which uses high temperatures placing samples inside an oven, although it could promote the hydrolysis and/or the matrix fusion, compromising the biocompatibility of some organs, similarly to autoclave, this sterilization method do not affect dental samples. An alternative method could be the chemical gas sterilization through ethylene oxide (EO), this method is very effective at low temperatures, however, the gas residues are alkylation agents, what can modify and/or compromise the properties of the biomaterial37, reason why the mechanical properties, appearance (color and shape) were checked before and after the procedures. Finally, the ionizing gamma irradiation (GAMMA), is an effective sterilization method, with perspective of also eliminate Sars-Cov2 with the highest potential for guaranteeing the safety of patients receiving skin, amniotic membranes and bone transplants34.The organ to be sterilized drives the sterilization method choice. The sterilization is a key step on biomaterials procedures, infections could cause rejection of scaffold or eventually the death of patientThe restoration of tooth loss is a great challenge in dentistry, and the search for a biological substitute envisages new therapeutic horizons. There are several mechanisms for demineralization, decellularization and sterilization of the organs, but was not found a definitive process for the dental scaffold. Dentistry aims not only on restoration but also on its regeneration, justifying the need of advancing on studies to mimic the teeth enabling it as biomaterial scaffold, what also will demand an efficient sterilization process.31, after partial demineralization-decellularization, presented remaining pulp on almost 30% of the samples. Would be possible to reach 100% of pulp removal, using higher concentration or time extension of exposition to demineralization solution, an alternative could be an endodontic intervention to access the pulp chamber, both avoided because they could interfere on the samples’ structural integrity. failure rates because of bacterial colonization are still high The existence of remaining pulp could provide conditions to cell proliferation38 and represent a considerable risk of rejection or inflammatory response39, reason why the sterilization methods were introduced in this phase of the study.In the first phase of this studyThe aim of this study was to obtain a viable dental scaffold, with preservation of structural properties, were compared commonly used sterilization methods (AS and DH) and high efficiency techniques (Gamma and EO), Mechanical resistance (micro-hardness), preservation of layers  under number 1529/2015. This study was conceptualized as primary, experimental, prospective, analytic and comparative design. Informed consent obtained from all patients and/or their legal guardian(s). In this design, 51 pre-molar teeth were obtained from healthy patients through orthodontic extraction procedure. The inclusion criteria for teeth selection were: healthy subjects without chronic use of any kind of drugs, radicular complete formation and signed written consent. Exclusion criteria were sectioned or bleached teeth, dental anomaly or dental whiteningPremolars were kept at room temperature in Falcon tubes at 10% formaldehyde solution before the use, then washed in de-ionized water, dried at room temperature (24 h). To make easier the access of cleaning substances to pulp chamber, teeth were emerged in a 500 ml of Phosphate Saline Buffer (PBS), with addition of 28 g of EDTA kept at 60C up to translucence (Ph4), then placed in individual recipients for 30 days at room temperature. Second step, were kept 30 days on 9% hydrogen peroxide to clean up the teeth, and final 30 days emerged in enzymatic detergent solution (Riozyme)/distillated water 50% each) to remove the biological residues. They were washed on deionized running water, dried at room temperature for 24 h and packaged in surgical grade paper to be sterilized.2. In two steps, the first 20 min sterilizing, and 30 min drying; EO (group GD3)—packed samples were sterilized through exposition to an ethylene-oxide (30%) + CO2 (70%) atmosphere at a relative humidity from 40 to 70%, pressure range 0.4 to 0.5 kgf/cm2, exposure time of 4 h with temperature range from 45 to 55 °C. Gamma (group GD5)—packed and sealed samples were exposed to radiation of 25 kGy with multipurpose irradiator of 60CO (CTR / IPEN / CNEN-SP), at room temperature and pressure.The first tooth was cut is 10 slices (~ 1.5 mm), washed, dried, packed and separated in pairs, these slices were intended to be grinded into powder for analisys at X-Ray Diffractometer (XRD) equipment. The 50 teeth + 10 slices were randomly separated into 5 groups of 10 + 2 slice each, named as groups for decellularization (GD) to avoid direct identification during analysis, and finally submitted to sterilization: DH (group GD1)—170 °C by 120 min; AS (group GD2)—from 60 to 129 °C, and pressure between 1.7 and 1.8 kgf/cmThe samples were characterized to confirm the preservation of ECM  and determine the efficacy of sterilization processes. The teeth were photographed  and radiographed in the longitudinal plane, before and after sterilization processes, using a positioning device to keep uniform distance. On radiographs were used an aluminum scale in order to standardize the different shades of gray observed, where the colorimeter Chroma Meter CR-400—Konica Minolta Camera Co., provided a quantitative measurement to evaluate the color variation after sterilization processes.10(PO4)6(OH)2 3(OH)), was decided to confirm the proportionality of Ca  and P (Phosphorus) in order to analyze if the sterilization treatments could cause a structural variation caused by temperature, pressure, exposition to ethylene oxide + CO2 or gamma irradiation. If existing, this variation could cause an atomic rearrangement crystallographic structure or adsorption of atoms from gas composition of controlled atmosphere (for Example on EO). The samples were ground into powder using pistil and mortar and measured in the XRD equipment D8 diffractometer, with linear position detector type LynxEye from Bruker. The powder was carefully positioned at sample-holder (PPMMA—Poly-methyl-methacrylate) and measured at room temperature under Cu kα radiation, wavelength 1 = 15,406 Å, 2θ scanning from 20° to 120°, step of 0.02° and exposition time of 1.2 s. The data were analyzed through Rietveld Method40 at Topas 4.2 software from Bruker AXS, and CIF  were obtained from ICSD . Mechanical Resistance—The samples were previously embedded in acrylic resin and polished, and then micro-hardness was measured at Shimadzu HMV 2 T equipment, using 490.3 mN, 0.05 load, and 10 s.To verify the micro-structural interfaces , the samples were carefully sliced, and analyzed at SEM equipment (EVO MA 15 Zeiss equipment), the samples were fixed on stubs and covered with exploded carbon tape, a thin carbon tape established the electrical contact between the sample surface and the sample-holder, allowing the images generation on SEM. The same equipment has an EDS probe included, where was possible to confirm the variation on atomic composition. An additional composition analysis was proceeded using an X-Ray Fluorescence (XRF) equipment, Bruker S2 Ranger, adjusted to analyze a 10 mm diameter and calibrated using pure Cu standard, the equipment has the limitation of analyzing the components through oxide, and Hydrogen is not detectable by the sensors. As the Hydroxyapatite formula is CaMicrobiological Test—To verify the viability of (anaerobic bacteria was used solution of Thioglycolate (THIO) and for aerobic bacteria, fungi and yeast, was chosen Tryptone Soya Broth (TSB). Negative control sterilization tests were proceeded using two sterilized tubes only with THIO and TSB, The positive tests were performed on 2 inoculated tubes, one only with THIO and the other with TSB solution with no teeth. From the control group GD4 (with no sterilization) one tooth was introduced in the tube with THIO, the second was placed into an inoculated tube with THIO, the third was introduced into a tube with TSB and the fourth into a tube with inoculated TSB. This procedure was done to warranty the cleaning substances residues are not enough to inhibit the microbial growth. The other groups had 2 teeth for each solution . All tubes were incubated at 37.5° C for 14 days. The microorganisms were Staphylococcus Aureus (ATCC6538), Bacilus subtilis (ATCC6633), Pseudomonas aeruginosa (ATCC 9027), Clostridium sporogenes (ATCC 11437), Candida albicans (ATCC 10231) and Aspergillus niger (ATCC16404). The concentration was 1 ml for each 15 ml of solution, with 10 to 100 viable microorganisms, in accord to Brazilian Pharmacopeia.Histology—The dental organs were included in resin block, sliced (2 elements of each group), polished and identified. The samples were colored with Hematoxiline Eosine (HE), Masson Goldner (MG) and Steven Blue (SB), and the pictures were obtained using Inverted Optical Microscopy Axiovert 40C (Carl Zeiss) through Axio Vision 4.1 software.The data were treated with Kruskal–Wallis (EDS), ANOVA 1 factor (Microhardness) and ANOVA double factor (mass weight losses) tests for statistical validation, in all cases the nullity hypothesis level was set at 0.05 or 5%.ECM preservation—The characterization techniques were used to confirm the preservation of ECM in terms of shape, atomic composition, diffractive information about atoms arrangement in the crystalline structure and the micro-hardness of the samples. The mass losses, optical images , absence of detected fractures, and minor changes on color suggest the different sterilization methods represents low impact on ECM, supposedly not compromising the scaffolds’ viability.As soon the sterilization processes were completed all the teeth were fractioned using sterilized cutting pliers, where was verified presence of pulp in 30% of the control group GD1) and also on Gamma group (GD5), 20% of DH (GD1) and EO (GD3), and 10% of AS (GD2). Observing SEM’s image, Fig.  and also41 , GD5 presented the expected hardness , GD2 (69.53 HVN), GD3 (70.61 HVN) and GD1 (77.55 HVN) these results, associated to statistical validation indicates the sterilization method interfere directly on the micro-hardness of teeth, it is important to confirm if this hardness increase can interfere on cell adhesion or not.All sterilization techniques caused significant elevation on hardness, where Gamma Radiation (GD5) is the least impacting, while the most impacting one was dry heat  do not change significantly, although presenting small intensity variation, mainly on angles range of 32–34 degrees; this variation is probably related to different region of sliced teeth analysis . The reminiscence of peaks on same angles indicates the structural arrangement was not modified, even after the sterilization processes. The refining parameters GOF (Goodness of Fit) 1.40 and weighted profile R-factor (RwP) = 8.51 reinforces the refinement is very close to expected HA’s data. On Fig. p = 0.009799, meaning a significant association between the masses losses and the method of sterilization.The “Table p = 0.026539 validate the hypothesis of different sterilization method interfere on the chemical composition. Dentine  test, with THIO and TSB solutions, confirmed there were no contamination on solutions or tubes. Two empty tubes were inoculated, and presented microorganisms proliferation, showing the effectiveness of THIO and TSB solutions. In sequence were run the positive control (PC) tests, preparing 20 falcon tubes, 4 for each sterilized group and control group . The absence of microorganisms, after incubation process, evidenced the efficacy of each sterilization method. Indeed, the control group (GD4) and the two empty tubes also were inoculated (PC and NC), presenting proliferation of microorganisms, validating the hypothesis the samples and solutions were not contaminated, the active actuation of THIO and TSB solutions and effectiveness of the sterility tests. The results are reported on Table 43 provided evidences about the efficacy of moist heat (autoclave) and dry heat sterilization procedures, being compatible to results obtained in this research. These two techniques are standardized on ISO17665-1:2006 and ISO 17665–2: for moist heat and ISO 20857:2010 for dry heat, even Ethylene Oxide (ISO 111135:2014) or irradiation (ISO 11137–2:2013) are standardized, but up to now, was not found reports to their application on dental teeth intended to be used as scaffolds.The use of sterilization procedures on medical or dental devices is a common practice, even on artificial implants as titanium teeth implants, the study of Manea44 reports the importance of sterilization when proceeding these tissues, some methods cited include moist heat (autoclaving), ethylene oxide and gamma irradiation. The study does not recommend ethylene oxide because of a toxic gas production (ethylene chlorohydrin) what could cause synovial inflammation, and focuses on gamma irradiation. Fölsch45 studied the thermo-disinfection on cancellous bones reporting the tensile strength was preserved, agreeing to supposed preservation of structural resistance on ECM.Femoral heads, processed bones, tendon or skin are good examples of allograft tissues used on transplantation or functional recovery surgeries, Singh31. When submitted to sterilization, the results suggest the influence of temperature and pressure variation on reduction of remaining pulp. Except by GD5 process, all the others presented a significant reduction; GD5 was run at room temperature (~ 20 °C) and pressure (~ 1kgf/cm2) still presented 30% of remaining pulp; GD1 at room pressure and 170 °C, reduced from 30 to 20%; GD3, at pressure between 0.4 and 0.5 kgf/cm2 and temperature 45–50 °C, also reduced from 30 to 20%; GD2, with pressure between 1.7 and 1.8 kgf/cm2 and temperature from 60 to 129 °C, reduced from 30 to 10%, indicating the combination of higher pressure and temperature have stronger impact on the reduction of remaining pulp.In this study, after partial decellularization process, almost 30% of samples still presented biological residues at the pulp chamber, repeating the results previously obtained on the partial demineralization studyAs the aim of this research was to evaluate the four sterilization methods, could be said the four methods were well succeeded, as all of them are suitable to produce sterilized scaffold. None of methods impacted on structural molecular arrangement (XRD), although, caused impact on chemical composition (XRF and EDS), and also caused higher micro-hardness. Considering the cost–benefit criteria associated to reduction of remaining pulp, the results suggest the Autoclave Sterilization (GD2) has some advantages in terms of competitive appeal. However, a deeper study to confirm the compatibility with stem cells should be proceeded previously to discard dry heat (GD1), ethylene oxide (GD3) or gamma radiation (GD5).The results evidenced the decellularization alone is not enough to eliminate microorganisms from dental scaffolds, and the four sterilization methods were fully effective with preservation of ECM molecular arrangements, but with some variation on chemical composition (mainly proportion of Ca/P). Gamma irradiation (GD5) and ethylene oxide (GD3) presents excellent results, but their viability are compromised by the costs and mainly the technology’s accessibility (requires very expensive equipment and/or consumables), exception to gamma irradiation (GD5), all the others also reduced the remaining pulp. Autoclave (GD2) presented easy equipment accessibility (with lower cost consumables), higher reduction of remaining pulp and complete sterilization, reason why was considered the most promising technique.


The cloacal anatomy is unique because the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, sampling for microbiome research can be difficult in birds, especially in agricultural production settings where it may not be feasible to sample the intestines, and cloacal swabs are often used. There is a need to evaluate laboratory methods for 16S rRNA sequencing in cloacal swab samples to ensure reproducible and trustworthy downstream results. We compared four DNA extraction methods from two commercially available magnetic-based DNA extraction kits. Mock communities and negative controls were included for each method and subjected to 16S rRNA sequencing. While extraction quality and yield differed between each extraction method, overall sequencing results were not affected, including alpha and beta diversity. Positive and negative controls are an important aspect of microbiome science and our findings lend guidance to future microbiome research in poultry.As the applications of microbiome science in agriculture expand, laboratory methods should be constantly evaluated to ensure optimization and reliability of downstream results. Most animal microbiome research uses fecal samples or rectal swabs for profiling the gut bacterial community; however, in birds, this is difficult given the unique anatomy of the cloaca where the fecal, urinary, and reproductive tracts converge into one orifice. Therefore, avian gut microbiomes are usually sampled from cloacal swabs, creating a need to evaluate sample preparation methods to optimize 16S sequencing. We compared four different DNA extraction methods from two commercially available kits on cloacal swabs from 10 adult commercial laying hens and included mock communities and negative controls, which were then subjected to 16S rRNA amplicon sequencing. Extracted DNA yield and quality, diversity analyses, and contaminants were assessed. Differences in DNA quality and quantity were observed, and all methods needed further purification for optimal sequencing, suggesting contaminants due to cloacal contents, method reagents, and/or environmental factors. However, no differences were observed in alpha or beta diversity between methods. Importantly, multiple bacterial contaminants were detected in each mock community and negative control, indicating the prevalence of laboratory and handling contamination as well as method-specific reagent contamination. We found that although the extraction methods resulted in different extraction quality and yield, overall sequencing results were not affected, and we did not identify any method that would be an inappropriate choice in extracting DNA from cloacal swabs for 16S rRNA sequencing. Overall, our results highlight the need for careful consideration of positive and negative controls in addition to DNA isolation method and lend guidance to future microbiome research in poultry. Rapidly advancing technology has made amplicon sequencing a popular and readily available method for analyzing microbiomes. As the field of microbiome science expands, so too does the need to standardize and optimize laboratory methods for best sequencing outcomes. There is growing interest in manipulating animal microbiomes to optimize production agricultural outcomes . For exaThe first step of 16S sequencing library preparation is to extract genomic DNA from the sample. Today, this is commonly performed with a commercial DNA extraction method that includes proprietary reagents and methodology. Past work has suggested that the choice of commercial DNA extraction method can affect DNA yield and quality ,9 or eveTo address this knowledge gap, we compared four different extraction protocols from two commercially available magnetic-based DNA extraction kits on cloacal swab samples collected from 10 hens. Positive and negative controls for each method were extracted and sequenced alongside the samples to enable comparison of method-specific contaminants. Extracted DNA quality and quantity were measured prior to library preparation and 16S sequencing. These data provide guidance towards appropriate methods for extraction of DNA from cloacal swabs for best 16S sequencing results in cloacal samples.All animal procedures described in this manuscript were approved by the Pennsylvania State University’s Institutional Animal Care and Use Committee (protocol no. PRAMS201647461). Female White Leghorn chickens (Hy-Line W36 strain) were reared in individual cages at the Poultry Education and Research Center of the Pennsylvania State University and exposed to a photoperiod of 16 h light and 8 h dark in a ventilated layer house kept at a standard 25–26 °C temperature . The animals were provided with layer mash diet and water ad libitum. At the time of sampling, the hens were approximately 2.8 years old. The animals were randomly subset from an ongoing study on metformin supplementation. Egg production records were maintained daily to determine ovulatory cycle patterns. Four cloacal samples from 10 adult White Leghorn chickens  were collected using a sterile swab  by inserting the swab ~1 inch into the cloaca and swirling for 2–3 s. All four swabs were introduced at once in the cloaca and swirled together, and all samples were collected on the same day. The swabs were immediately placed into sterile microtubes, transported to the laboratory on ice, and stored at −80 °C until DNA extraction.g for 30 min to pellet bacterial cells and debris. Finally, each pellet was suspended in 300 µL of PBS and homogenized again for 30 min in the same conditions described above. All samples were kept at −80 °C until processing.Before DNA extraction, all swab samples were randomized and submitted to a pre-processing step. Briefly, one milliliter of sterile phosphate buffer saline solution  was added to each tube, which were homogenized at 20 Hz for 30 min in the Bead-Ruptor96 . Samples were then transferred to a new tube and centrifuged at 14,000× DNA extraction was performed in a UV-sterilized biosafety cabinet, and pipettes and other equipment were sterilized with 70% ethanol in between each extraction. Genomic DNA was extracted from cloacal samples, a mock community , and negative controls (buffers) using two different DNA extraction methods and four different protocols: MagMAX™ CORE mechanical lysis module and MagMAX™ CORE nucleic acid purification method , MagMAX™ microbiome ultra nucleic acid isolation method , in-house bead loading system and MagMAX™ CORE nucleic acid purification method using clarifying solution , and in-house bead loading system and MagMAX™ CORE nucleic acid purification method using Reagent DX . For Method 1 and Method 2, DNA extraction was performed according to the manufacturer’s instructions. For Method 3 and Method 4, DNA extraction was performed according to the manufacturer’s instructions, except for the use of zirconia bead tubes , within which samples were lysed before they were added to the sample plate. Briefly, 450 μL of lysis buffer were added to each zirconia bead tube corresponding to a sample. The lysis solution was prepared with the lysis buffer included in the methods and 2.05 μL/reaction of clarifying solution  or Reagent DX  for Method 3 and Method 4 protocols, respectively. Subsequently, 200 μL of each sample were transferred to the zirconia tubes, which were homogenized in the Bead-Ruptor96  at 20 Hz for 2.5 min. Samples rested for 5 min and were homogenized for an additional 2.5 min. Zirconia tubes were then centrifuged at 2500 rpm for five minutes, and 500 μL of the lysate from zirconia tubes were transferred to the sample plate, and each protocol proceeded according to the manufacturer’s instructions.Extracted DNA quantity was measured fluorometrically with Qubit  at the Pennsylvania State University Genomics Core Facility and spectrophotometrically with a NanoDrop . The extracted DNA was stored at −80 °C until further library preparation.YCAGCMGCCGCGGTAA; REV: GGACTACNVGGGTWTCTAAT, following Earth Microbiome Project protocols [Polymerase chain reaction was performed on genomic DNA with the standard 515F and 816R primers- FWD: GTGrotocols ,13. The https://www.arb-silva.de/documentation/release-1381/, accessed on 1 February 2021). Data were rarefied based on the sample with the lowest sequence count using the “rarefy_even_depth” tool of the R package phyloseq v 1.34.0 and used for diversity analysis [Reads were processed using the dada2 (v. 1.18.0) R package . Raw reads were quality-filtered using the “filterAndTrim” option. Twelve different parameters were tested to optimize the quality-filtered reads  based onanalysis . Data weAs the animals sampled were a subset from a larger study where they were supplemented with medicated feed, the data were assessed to ensure the data from individual chickens were alike enough to be considered biological replicates. Data from all four methods were combined and Kruskal–Wallis was performed for the variables of DNA quantity (ng/µL), A260/280, and A260/230 ratios between chickens. No significant difference was found, so individual chickens were treated as replicates. All variables were assessed for normality by plotting a histogram and performing Shapiro–Wilks’s test , followip values were adjusted for multiple comparisons with Bonferroni’s correction. After adjustment, significant results were subjected to Dunn’s post hoc test. All statistical analyses were performed in R version 4.0.1, and raw data (NanoDrop and Qubit data) and R scripts are available at https://github.com/gandalab/bird-dna-extraction. Sequencing reads are available at the NCBI Sequence Read Archive under accession number PRJNA720437.Alpha diversity metrics (Shannon’s index and observed taxa) were calculated with the phyloseq package and visualized with the ggplot package ,18. Brayp = 0.021) and Method 2 , and Method 4 had higher DNA quantity than both Method 1  and Method 2 . There were no differences between Method 3 and Method 4  or between Method 1 and Method 2 .The quantity of extracted DNA, measured through fluorescence with a Qubit instrument, differed between extraction methods. Method 3 and Method 4 had higher extraction yield than both Method 1 and Method 2 . Specifip = 0.037) and Method 4  differed significantly from the ideal ratio, while Method 1  and Method 3  did not (The ratio of A260 to A280 absorbances (A260/280) are used to assess the purity extracted DNA as nucleic acids absorb at 260 nm and proteins absorb at 280 nm . Median  did not b.p = 0.0056), Method 2 , Method 3 , and Method 4  all had strong differences from the ideal ratio. However, after PCR amplification and AmPure magnetic bead cleaning, the ratios were recovered to acceptable values before sequencing, and only Method 2 had significant differences from the ideal 2.00 value (The ratio of A260 to A230 absorbances (A260/230) are considered a broad category for detecting contaminants, including phenols. Median A260/230 values for each method are reported in 00 value .n = 139,423) and positive controls  were 6.34 and 5.91 times higher than negative controls , respectively. The negative controls in all four methods had detectable bacterial biomass that was reflected in the respective counts of non-chimeric reads .Sequencing of the 16S rRNA V4 hypervariable region was carried out in 40 samples using the Illumina MiSeq platform. A total of 6,595,579 reads were obtained from cloacal swab samples, with a mean read count of 196,889 per sample and a mean read count of 1,648,895 per DNA extraction method . Raw sequencing data were visualized in MultiQC to determine filtering and trimming parameters . FilteriTo enable comparison of method-specific contaminants, a negative (extraction buffer blank) and positive (known mock community standard) control were extracted, amplified, and sequenced for each commercial magnetic-based extraction method. The mock community composition of each method was compared to the expected values given by the mock community standard manufacturer. All methods showed similar composition with minimal detected contaminants in the positive controls and recapitulated the expected composition of the mock community a.Asinibacterium and Pseudomonas were the most abundant genera detected in the NC of Method 1 and Method 2. Method 3 also had Asinibacterium as the most abundant genus, followed by unclassified taxa. Burkholderia-Caballeronia-Paraburkholderia and Lactobacillus dominated the bacterial composition of NC in Method 2. NMDS visualization showed that the negative controls of Method 1 and Method 3 clustered together and separately from the samples, while the negative control of Method 4 clustered closely to the cloacal samples , which underwent library preparation and sequencing. Reads obtained from each NC were derived from 21 (Method 1), 36 (Method 2), 26 (Method 3), and 77 (Method 4) genera. The contaminant profiles of Method 1 and Method 3 NC were similar, but Method 2 and Method 4 were vastly different b. Asinib samples a.p values for multiple comparisons; Proteobacteria had the lowest relative abundance in Method 4 compared to Method 1 , Method 2 , and Method 3 . The genus Lactobacillus was the most abundant taxa, followed by Actinomyces, Bacteroides, Romboutsia, Enterococcus, Corynebacterium, and Gallibacterium, and Lactobacillus, . Non-multidimensional scaling based on Bray–Curtis distances was performed to evaluate the impact of extraction method on microbial community composition (p > 0.05), and therefore, beta diversity was tested with permutational ANOVA. Permutational ANOVA was performed both with and without the negative and positive controls, and no significant differences were detected between DNA extraction methods . Similar results were observed in the principal coordinate analysis (PCoA) based on weighted Unifrac distance   .Four commercial DNA extraction methods were tested on 10 cloacal samples from White Leghorn hens to assess if the extraction method affected library preparation or 16S sequencing results. We found that although the extracted DNA quality and quantity differed between methods, overall 16S sequencing results  were not affected, and only one phylum had differences in relative abundances between methods. Methods 3 and 4 (both protocols with the in-house bead loading system) resulted in higher DNA extraction yield with similar quality.Differences in DNA yield among extraction methods had no effects on microbial diversity. This result is in accordance with studies using human feces and pig feces and hospital sewage ,21. InteLactobacillus, which has been previously described in young broiler chicks [Pseudomonas and Romboutsia are the most abundant genera in laying hen cloacal swabs [Taxonomic analysis of cloacal samples showed similar composition at phylum and genus level despite extraction method and were similar to previous work in poultry. One phyla, Proteobacteria, was found to be significantly less relatively abundant in Method 4 than the three other extraction methods; however, there were no differences in genera. Firmicutes, Proteobacteria, and Actinobacteria have been found to be the most abundant phyla in cloacal samples in broiler and laying hens ,23,24,25al swabs ,23. The al swabs . Since wFaecalicoccus and Enterobacteriaceae. With only one control per method, statistical testing was not possible, and there were only slight numerical differences in the relative abundance values for each bacterial strain compared to the expected mock community. Interestingly, Lactobacillus and Escherichia-Shigella were both also detected in the negative controls of all four methods, which suggests that these bacteria may be present as mock community standards as well as contaminants. In Method 2 and Method 4, Lactobacillus made up a relatively large percentage (~10%) of the total relative abundance of the negative controls.Mock communities and negative controls were also evaluated for each extraction method. The four positive controls generally agreed with the expected mock community values. However, it is important to note that contaminant genera were detected in all four positive controls that were not part of the expected mock community makeup, which were composed of Asinibacterium was the most common genera found in Methods 1, 3, and 4, while most contaminants in Method 2 were comprised of the least abundant genera  although each method was compared in the same 10 animals, eliminating a potential source of intra-animal variability. Additionally, four methods from two commercially available kits were measured, and many more commercially available options exist for DNA extraction. Future research should compare additional methods  to determine if those affect sequencing outcomes. The negative and positive controls were added at the extraction steps, and there were no unused swabs as an environmental negative control, so it is not possible to determine if any bacteria in the cloacal samples were environmental contaminants.This study is limited by the relatively small sample size  had the highest extraction DNA yield and comparable extraction quality, the Method using Reagent DX (Method 4) resulted in the highest number of contaminant genera detected in the negative control and the lowest relative abundance of Proteobacteria in the cloacal swab samples. Therefore, it may be most appropriate to use clarifying solution in place of Reagent DX for DNA extraction in cloacal swabs. As no differences were observed in bacterial diversity metrics, we can conclude that the choice of specific DNA extraction method tested in this study does not affect overall sequencing outcomes in cloacal swabs; however, it is critically important to add both positive and negative extraction controls to microbiome projects in order to properly detect and account for contaminant bacteria. This research provides guidance to investigators in future studies on animal microbiomes, particularly those involving poultry cloacal samples.


Treponema, Mycoplasma, Porphyromonas, and Fusobacterium were consistently identified in the majority of DD lesions, and were the best genera at differentiating DD lesions from normal skin. Individual study and 16S hypervariable region sequenced had significant influence on final DD lesion microbial composition (P < 0.05). These findings indicate that members of Treponema, Mycoplasma, Porphyromonas, and/or Fusobacterium may have significant roles in DD pathogenesis, and should be studied further in respect to elucidating DD etiopathogenic mechanisms and developing more effective treatment and mitigation strategies.Bovine digital dermatitis (DD) is a significant cause of infectious lameness and economic losses in cattle production across the world. There is a lack of a consensus across different 16S metagenomic studies on DD-associated bacteria that may be potential pathogens of the disease. The goal of this meta-analysis was to identify a consistent group of DD-associated bacteria in individual DD lesions across studies, regardless of experimental design choices including sample collection and preparation, hypervariable region sequenced, and sequencing platform. A total of 6 studies were included in this meta-analysis. Raw sequences and metadata were identified on the NCBI sequence read archive and European nucleotide archive. Bacterial community structures were investigated between normal skin and DD skin samples. Random forest models were generated to classify DD status based on microbial composition, and to identify taxa that best differentiate DD status. Among all samples, members of  Bovine digital dermatitis (DD) is an infectious skin lesion that was first reported in Italy in 1974 , and is Treponema as the main pathogen of these lesions. More recent evidence shifts the focus to studying the roles of both Treponema and additional anaerobic bacteria in lesion formation and progression, leading to a polymicrobial hypothesis of DD causation .Searches for publicly available data were performed on the NCBI Sequence Read Archive (SRA) database and the European Nucleotide Archive using the search term “digital dermatitis”, and limiting search results by “BioProject”. BioProject accession numbers (study identifiers) containing high throughput sequencing reads and associated metadata were collected. For inclusion in the meta-analysis, all studies had to have publicly available sequencing reads before September 1, 2020. Studies were required to be a bovine DD metagenomic study, and have conducted deep amplicon sequencing of a 16S rRNA hypervariable region using universal bacterial primers on samples originating from bovine skin. Raw data  and sufficient metadata (containing information differentiating samples by DD diagnosis) had to be publicly available for inclusion in the meta-analysis. Additional information on sample collection was obtained for each study. All samples were collected with biopsy punches from feet cleaned with water or chlorhexidine of either live animals or at a slaughterhouse. Depth and exact location of skin lesion samples were not universally available across all studies. Other relevant details concerning experimental processing of samples before sequencing are described in Treponema-specific V3V4 16S rRNA regions were processed with Bowtie2 v.2.3.5.1  test determined any significant differences of diversity between DD statuses. For beta diversity analyses, ASVs were grouped at phylum, family, and genus taxonomic levels, and Bray-Curtis dissimilarities between samples were explored using Principal Coordinates (PCoA) analysis. Permutational ANOVA (PERMANOVA) with 999 permutations, followed by a test for multivariate homogeneity of group dispersions (BETADISP) was performed to identify differences in microbial composition between DD negative and DD positive samples.Due to the lack of consistent information across studies on individual lesion stages, all samples included in analysis were categorized as “DD positive” or “DD negative”. DD positive samples were defined as any sample from DD lesion skin at any stage of the disease. DD negative samples were classified as any sample from skin with no visible DD lesion present. Any samples that were obtained from treated lesions were removed from downstream analysis. For diversity analyses, samples with less than 1500 reads were removed, and the remaining samples were rarefied down to the minimum sequencing depth. Observed ASV count, Chwfi 2ao1 richness estimate, Fisher’s alpha and Shannon’s diversity index were used to evaluate diversity (richness and evenness) within DD positive and negative samples. Analysis of variance (ANOVA) followed by The influence of different study variables and experimental designs on microbial composition was measured exclusively on DD positive samples by repeating the diversity analysis methods described above. Alpha diversity and relative abundance plots evaluated the effect of different 16S rRNA hypervariable regions sequenced, and beta diversity measured the variation in microbial composition by individual study, 16S rRNA hypervariable region, and sequencing platform. All diversity metrics and statistical analyses were conducted using the vegan package (v. 2.5.6) in R .P value of less than 0.05 was considered statistically significant for all comparisons. All analysis and figures were completed in R version 3.5.3.Random forest modeling was used to predict DD status based on microbial composition, and identify the microbiota that best differentiates DD positive from DD negative samples. Prior to modeling, ASVs were grouped at phylum, family, and genus ranks and rare taxa (present in less than 1% of samples) were removed. Sample ASV counts were normalized using a centered log ratio transformation, with a pseudocount of 1 applied to all ASVs. Randomly generated training sample sets were made of 70% of each DD negative and positive samples, and testing sample sets contained the remaining 30% of samples in each group. Random forest models containing 1000 trees were trained using 100 repeats of 10-fold cross validation, and the number of variables sampled at each node was optimized as part of the caret R package v.6.0.86 . Model pA total of 24 BioProject IDs were identified upon the initial search. Next, 12 studies were removed because they did not conduct deep amplicon sequencing of a 16S rRNA hypervariable region. Four studies were excluded because they were not from bovine skin/lesion samples, or did not have any raw fastq files associated with the BioProject ID. One study was removed because it did not use universal bacterial primers for sequencing. Finally, one study was removed due to a lack of metadata to differentiate samples by DD status. Included in the meta-analysis were 6 studies Table 1,(P < 0.05) diversity, richness, and evenness when compared to DD negative skin microbiota ((P < 0.05) in microbial composition between DD negative and DD positive skin ((P < 0.05) at the phylum level  as identified in Permutational analysis of variance on Bray-Curtis distances identified BioProject  as the largest source of variation (20%) in microbial composition between DD positive samples, regardless of taxonomic levels tested Table 5.(P < 0.05) in richness, evenness, and diversity within DD positive samples from the V3V4 region compared to both V1V2 and V1V3 for all diversity measures evaluated ; therefore, interest should remain on Fusobacterium as a potential pathogenic agent of DD. Porphyromonas is also a possible contributor to DD pathogenesis that is detectable in all active lesions in one study , but along with Fusobacterium, its superficial location in the dermis has led some studies to limit their conclusions on its potential involvement  the samples that used V3V4 sequencing were also the only samples to include skin samples from beef cattle breeds rather than just dairy breeds; 2) V3V4 primers may amplify a wider range of bovine DD skin microbiota. DD in beef cattle is a recent field of study, as DD is emerging in those populations , the Canadian Dairy Commission under the Workplace Development Initiative, and the Simpson Ranch Grant at the University of Calgary Faculty of Veterinary Medicine.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.