Leg extension exercise increases the quadriceps muscles’ blood flow 100-times; transporting 17 sextillion O2 molecules per kilogram per minute from microcirculation (capillaries) to mitochondria powering their cellular energetics. Within these muscles, the capillary network constitutes a prodigious blood-tissue interface essential to exchange O2 and carbon dioxide requisite for muscle function. In disease, microcirculatory dysfunction underlies the pathophysiology of heart failure, diabetes, hypertension, pulmonary disease, sepsis, stroke and senile dementia. Effective therapeutic countermeasure design demands knowledge of microvascular/capillary function in health to recognize and combat pathological dysfunction. Dated concepts of skeletal muscle capillary (from the Latin capillus meaning ‘hair’) function prevail despite rigorous data-supported contemporary models; hindering progress in the field for future and current students, researchers and clinicians. Following closely the 100th anniversary of August Krogh’s 1920 Nobel Prize for capillary function this Evidence Review presents an anatomical and physiological development of this dynamic field: Constructing a scientifically defensible platform for our current understanding of microcirculatory physiological function in supporting blood-mitochondrial O2 transport. New developments include: 1. Putative roles of red blood cell aquaporin and rhesus channels in determining tissue O2 diffusion. 2. Recent discoveries regarding intramyocyte O2 transport. 3. Developing a comprehensive capillary functional model for muscle O2 delivery-to-V̇O2 matching. 4. Use of kinetics analysis to discriminate control mechanisms from collateral or pathological phenomena.When exercising humans increase their oxygen uptake (V̇O Breaking World athletics records and achieving monumental physical milestones  captivates public attention whilst extending the proven boundaries of physical performance. Development of novel methods  combined with high fidelity measurements  mathematical and kinetics modeling approaches have better resolved the physiology of elite performance in health and also our understanding of the exercise intolerance that erodes life quality and predicates morbidity and mortality in disease.2 delivery (Q̇O2) and its instantaneous matching to V̇O2. We are far closer to understanding what the key functional parameters of sustained muscular performance are—V̇O2 kinetics, critical power , and the maximum V̇O2 (V̇O2max)  . A compe2 that is Q̇O2 dependent (see 13), during exercise V̇O2 and blood flow (Q̇) should increase in a ratio of ∼1:5 or 6,2 content should fall  as a hyperbolic function of increasing V̇O2.16 Moreover, when measured within the skeletal muscle, V̇O2 should increase with little delay in an exponential fashion with a time constant (τ) of some 10 to 40 s in young, healthy individuals.; rev.1Crucially, any model of muscle microcirculatory control and function must cohere with and support these known facets of oxidative performance. Specifically, resting muscle preparations cannot have a V̇O2 kinetics) or quantitatively , and is unnecessary to support the dynamic control of microcirculatory Q̇O2 and V̇O2 kinetics.The most pervasive capillary control models over the past century are based upon August Krogh’s Nobel prizewinning “capillary motor” schema.2 delivery or closed), our understanding of the transport of O2 from RBC to mitochondria has undergone substantive revision based upon recent discoveries. For instance, the diffusivity of O2 across its “final frontier” is likely dependent, in part, upon: (1) Newly discovered channels in the RBC membrane. (2) Recruitment of capillary exchange surface along the capillary length . (3) Network interdependence of O2 exchange among adjacent microvessels. (4) More homogeneous O2 pressures in the interstitial space surrounding myocytes. (5) O2 tracking along sarcolemmal and intracellular membranes, including interconnected mitochondria. (6) Interplay between deoxygenated myoglobin (Mb) and nitric oxide (NO) release to control cytochrome oxidase activity and better spatially coordinate mitochondrial ATP production and O2 availability. These considerations are important for construction of a contemporary model of capillary function and O2 transport: One that has far less reliance on control at the individual capillary level and, at least in health, minimizes the importance of capillary–mitochondrial diffusion distances per se.Moreover, beyond the Kroghian binary concept of capillaries , whose Commentary on Anatomy in Avicenna’s Canon became known to the Western world only in the early 20th century, he may have reconsidered his “porosities of the flesh” concept. As translated from “Commentaries…” by Meyerhof28” “…there is no passage between these two cavities [right and left ventricles]; for the substance of the heart is solid…” and “….the penetration of the blood into the left ventricle is from the lung…” leading Ibn al-Nafis to conclude that there must be small communications between the pulmonary artery and vein. See West,29 for a reproduction of the original Arabic text opposing the presence of septal connections between the ventricles from Meyerhof.28 Thus, at least as early as the 13th century in the Arab world the pores of Galen in the interventricular septum had been falsified. In Europe, Michael Servetus (1511 to 1553), who, West29 speculates, may have been aware of Ibn al-Nafis’s work—though historians consider this unlikely—wrote that the blood passed from the pulmonary artery through the lung to the left ventricle and became “reddish-yellow” in the process.30; see also29 As a mark of Harvey’s intuitive brilliance, a few years before his death in 1657, he experimented with the pulmonary circulation of a throttled man. After ligating certain vessels he demonstrated that water flowed easily from the pulmonary artery through the lungs and into the left ventricle.31; rev.29 A finding that, for the emerging generations of microscopists, identified the lungs as an organ where visualization of the smallest vessels connecting the arterial and venous circulation might be possible in vivo.When William Harvey’s De Motu, in Crevalcore near Bologna. Malpighi used both single magnifying lenses and a compound double-convex lens microscope and observed capillaries in the alveolar walls of the frog lung.32 He described the capillaries as “tortuous” and clarified conclusively that blood “is always passed through tubules” remaining within the vessels and not “poured out into spaces” (translation from33). Whereas, Jan Swammerdam (1637 to 1680), had described RBCs or corpuscles three years earlier;34, see also35 Malpighi mentioned these only as “particles” within the blood, probably because he could not determine their color.36Enter Marcello Malpighi who was born serendipitously in 1628, the year Harvey published 38 Hales estimated the pulmonary capillary diameter to be around 17 µm  with pulsatile flow and a RBC transit time just over 2 s. Capillaries were distinguished from other blood vessels by Marshall Hall (1790 to 1857) as being of “…an intermediate station…” interposed between the “…last branches of the arterial system and the first roots of the venous…” 39, p.18 He noted that, within capillaries compared with upstream vessels, blood velocity was halved and intuited that “…a more diffused and slower circulation is required for administering to the nutrient…functions” and considered capillaries to be of “uniform character and dimensions.”39, pp.the same diameter throughout”40 but the field was obscured with confusing terms such as “capillary arteries,” “capillary veins”, and “minute vessels.” 41 Improvements in light microscopy toward the middle of the 19th century revealed the cell as the primary structural base of tissues. 43; rev.44 This realization and Schwann’s “cell theory” were focal to the recognition of the capillary wall or endothelium.Stephen Hales (1677 to 1761), minister of Teddington parish near London, may have been the first person to coin the term “capillary.”blood in the finest capillaries…flows…in simple furrows, or canals, whose walls are formed by the surrounding cellular tissue.” 45, p.8 At that time it was considered impossible for “the processes of nutrition and absorption being carried on through the coats of vessels.”42 Counter to this notion was that capillaries in the ears of reptiles and birds when injected with dyes were distinct from adjacent tissues—evidencing “parieties” or walls.46 It was left to Theodor Schwann (1810 to 1882), in 1839 to identify what would later be called the endothelium in the tails of tadpoles.43 However, adherents to each side of the argument, walls versus no walls, engaged in strident debate based upon assumed properties of such a barrier and the presumption that it would prevent the known blood-tissue transit of, for example, leukocytes (see ref.44 for a stimulating account of the controversy). This despite Augustus Waller’s (1814 to 1870) demonstration, in real time, of leukocytes “protruding” from vessels at the site of inflammation in his frog’s tongue intravital microscopy preparation.47 A process called diapedesis.Reminiscent of Harvey, J.W. Earle in 1835 portrayed capillaries as membrane-less channels such that: “…48 This set the stage for addressing how capillaries subserve their cardinal responsibility for facilitating blood–tissue exchange. Ernest Henry Starling (1866 to 1927) would take up the challenge for substrate and fluid exchange, whereas Schack August Steenberg Krogh’s (1874 to 1949) focus was O2 delivery, primarily in skeletal muscle.At the close of the 19th century Julius Cohnheim (1839 to 1884), a student of Rudolf Virchow (1821 to 1902), had demonstrated that the capillary wall was a living organ rather than simply an inert membrane.closed” in resting muscle and opened or “recruited” at the onset of contractions. (B) Each capillary representing an independent fixed unit of O2 delivery with intracapillary diffusion distances determining the efficacy of mitochondrial O2 delivery. Despite these notions being resoundingly falsified, they remain at the forefront of undergraduate and medical school teaching and exert a persistent influence over the microcirculatory control field today.Since Krogh’s 1920 Nobel prize, the dominant concepts of capillary function, especially within skeletal muscle, have been driven by Krogh’s work and theories.everybody talks about it but nobody reads it” to paraphrase the great Mark Twain.49 For instance, one of the most memorable and enduring “facts” regarding the human circulation is that the total length of microvessels in skeletal muscle (or the human body) is 100 000 km or more; sufficient to encircle the Earth at the equator about 2.5 times. This figure comes initially from Krogh’s book,25, p.10 where capillarity values from guinea pigs are inappropriately applied to human muscles, and is grossly in error. Our best estimates from contemporary data yield a total length of some 9 000 to 19 000 km; less memorable, perhaps, but more accurate and supporting a very different regulation of capillary structure and function in health and disease. Specifically, where the blood volume necessary to fully perfuse the muscle capillary bed is only 2% to 3% of total blood volume versus over 30% (Krogh’s estimate). In the latter instance, if correct, poorly regulated (muscle) capillary opening and perfusion could compromise cardiovascular regulation and homeostasis. In many ways, the microcirculation today is treated as a classic—“Pervasive Myths and The Microcirculation) presents some of the major time-honored beliefs originating from Krogh;50 and, in opposition, the more recent evidence that informs and shapes a contemporary understanding of capillary function and blood-mitochondrial O2 transport matching structure and function to physiology  most capillaries are closed and do not support RBC or plasma flux. At the start of contractions, these capillaries are recruited and support RBC flux. Therefore an increase in the number of RBC-flowing capillaries is requisite to support V̇O.2, hematocrit) O2 transport principally occurs via elevated RBC flux, velocity and hematocrit in already-flowing capillaries  V̇O2], longitudinal recruitment, network effects), and myocyte-associated processes . These latter processes are considered below (see the section “Intramyocyte O2 Transport: Sarcolemma-Mitochondria”).When circulation-intact resting muscles are visualized by intravital microscopy under physiological conditions  the vast majority  of capillaries support RBC flux  have been demonstrated in the absence of any increase in the numbers of RBC-perfused capillaries.89 This profile matches closely that measured for pulmonary17; rev.1 and leg muscles112 V̇O2 in humans during voluntary exercise.Using a combination of intravital microscopy and phosphorescence quenching in the rat spinotrapezius muscle, rapid, and physiological V̇O113 nor impact the fundamental proportionality between muscle V̇O2 and Q̇.16 (B) Using endothelial dye perfusions essentially all capillaries in muscles of anesthetized and conscious rats at rest evidence flow (at least plasma) nullifying the opportunity for de novo recruitment.80 (C) Broadly accepted values for resting muscle blood flows are compatible with 80% to 90% of total capillaries sustaining flow with >10 RBCs/s.89,, rev.119, A valid concern reflects that, because direct observation of the skeletal muscle capillary bed requires that the animal be anesthetized, the presence of RBC flux in most capillaries may be an artifact of anesthesia or surgery. However, several lines of evidence support that neither anesthesia nor surgery perturb the underlying physiology in this regard. Specifically: (A) Surgical exteriorization does not alter muscle Q̇127 based upon some of the highest muscle blood flows measured in human knee-extensors, support that capillary RBC mean transit times of only ∼0.1 s can facilitate fractional O2 extractions as high as 80%.One theoretical argument advanced to support that capillary recruitment must occur from rest-exercise is that this is necessary to limit the fall in capillary RBC transit times. However, this notion is specious because, irrespective of how you get there—recruitment or simply increased flux in already flowing capillaries—RBC transit time during maximal exercise will be exactly the same as it is determined by the ratio of capillary volume/blood flow. Moreover, the estimations of Richardson and colleagues,Myth 2:Capillaries have a binary function: Either closed or open (see2 delivery#2) with the latter supporting a fixed capacity  for RBC flux and O.2) and diffusive (DO2) capacity of individual capillaries  that are far higher than intramyocyte (PimO2) PO2’s  and O2 diffusion among different vessels in the microcirculatory network,137 the latest O2 transport models advocate for dependent tissue volumes that decrease rather than increase (see Krogh–Erlang model) with distance from the O2 source—be that the capillaries or interstitial space  decreases systematically with distance from the capillary generating anoxic loci in resting muscle beyond the boundary of the “Krogh cylinder” (According to the Krogh–Erlang model O#4). If true this places an obligatory limitation on capillary–mitochondrial diffusion distances.2 (PmvO2), PisO2 and PimO2 at rest and during contractions, not technically feasible in Krogh’s time, have revealed very different profiles than hypothesized by the Krogh–Erlang model  and human quadricepsisO2 to PimO2) PO2 gradient during contractions. Although possibly a limitation of the cryomicrospectroscopy and proton NMRS measurement techniques neither radial nor longitudinal intracellular PimO2 gradients or anoxic loci  have been detected. If intramyocyte PO2 gradients do exist, because of the overall low PimO2, they must be small. These data support the notion that intramyocyte O2 transport is extremely effective and therefore that diffusion distances from sarcolemma to even the most distant part of the mitochondrial reticulum are not limiting .Measurements of microvascular POodel cf.  #7. SpecMyth 5:RBC and plasma flow through capillaries are controlled by so called precapillary sphincters.capillary motor regulating mechanism”144 that won him the Nobel prize was central to the capillary recruitment hypothesis and emplaced capillaries themselves in control of muscle Q̇ . Throughout the 1930s capillary contractility was repeatedly challenged with advocates150, 151 and opponentsKrogh’s “uscle Q̇  #5. Krog50 Here the plot thickens. In the frog mesentery in 1937 Zweifach finds no precapillary sphincters,155 but in 1939 he found such in both mouse and rabbit mesentery.156 In 1942 Chambers and Zweifach produced motion pictures evincing “sphincter like functioning of the precapillaries at their junction with the arteriole.”157; see also Sakai and Hosoyamada.158 Despite Zweifach’s observations of precapillary sphincters solely in the mesentery and not in all species, within a decade, they were adopted as the presumptive site of capillary hemodynamic control within ALL microcirculations in physiological and medical textbooks.; rev.158 Also disturbing, and a clear case of confirmation bias, was the propensity for researchers, from that point on, to attribute changes in vascular resistance to “precapillary sphincter tone” without any evidence for such.158 did not identify these structures in any tissue.In the absence of satisfactory evidence that either the capillary endothelium or pericytes could occlude the capillary lumen Benjamin Zweifach’s attention shifted to the beginning of the capillary, where the capillary branch departs from the arteriole. At that specific location he detected a narrowing which he termed a precapillary sphincter.ATP channel blockade with glibenclamide and other conditions that decrease blood flow to skeletal muscles do so via arteriolar vasoconstriction and there is no evidence that RBC flux can be halted by active luminal constriction specifically in individual capillaries.169; rev.170Given that absence of evidence is not necessarily evidence of absence with respect to structure it is also telling that presence of precapillary sphincters has not been demonstrated physiologically. For instance, hyperoxia, KMyth 6:2 stores and produce vasoactive metabolites which then vasodilate arterioles and “open” up previously closed capillariesFollowing the onset of contractions it is necessary for muscles to deplete their O.2, ultrasound, NMR, and mathematical modeling have permitted resolution of rapid muscle metabolic and hemodynamic transient responses following exercise onset. These techniques have helped unveil the mechanistic bases for vascular control dynamics.Advances in intravital microscopy, phosphorescence quenching measurement of POsleeping giant” in that uncontrolled dilation would crash blood pressure catastrophically.171 Specifically, if muscle vasodilation occurred prior to cardiac output increasing, blood pressure would plummet. By the same token delayed muscle vasodilation would produce a potentially dangerous blood pressure spike. In young healthy subjects, neither extreme response is seen. Rather, following the onset of rhythmic cycling exercise mean arterial pressure rises modestly with response kinetics that are far slower than that of muscle Q̇ :172 Supporting a close dynamic matching between the time course of both processes.Logically, if the cardiovascular system is to subserve its primary function of blood pressure control, there must be a tight coordination between central cardiovascular (cardiac output) and muscle Q̇ dynamics. Skeletal muscle has such a great capacity for vasodilation that it has been described as the “173 Later Krogh and Lindhard in 1913,174 measured correspondingly fast heart rate kinetics at the onset of cycle exercise in humans and Anrep and von Saalfeld in 1935 showed that muscle hyperemia was initiated simultaneously with the onset of contractions.175 Despite this evidence, as late as the close of the 20th Century, it was widely considered that, following the onset of exercise, “muscle” PO2 plummeted toward zero and subsequent vasodilation was dependent upon production of metabolites and sympathetic vasodilator activity.177As early as 1895 the eminent Swedish physiologist Erik Johan Johansson (1862 to 1938) demonstrated that electrical stimulation of the legs of rabbits increased heart rate within 0.5 s.178; rev.179 Moreover, the obligatory role of the muscle pump181 in initiating the rapid hyperemia has been challenged by experiments supporting that contraction-induced vascular deformation induces an almost immediate vasodilation183 possibly involving integrins184 and mechanosensitive ion channels.185 Importantly, Behnke and Delp186 showed that isolated rat skeletal muscle arterioles do evince the requisite fast relaxation dynamics in response to elevated flow, acetyl choline and NO. However, Clifford and Tschakovsky,179 argued that a system which elevates muscle19,89 Q̇ within 1 s after a contraction is unlikely to depend on diffusion of some soluble vasoactive mediator. This conclusion would also be relevant to the extent to which sympatholysis189 could participate in the initial Q̇ response.190Today it is recognized that mechanisms initiating the increase of muscle Q̇ may be very different from those that sustain its exercising steady state.191 and capillary192 Q̇ increases as fast or faster than mitochondrial V̇O2 such that, for the first 10 to 20 s of contractions, muscle PmvO2 and venous O2 content do not fall and may even rise .Notwithstanding its precise mechanistic bases, following the onset of muscle contractions, in both exercising human muscleven rise . 19,20,8Myth 7:2 diffusing capacity (DO2) can only increase from rest to exercise by recruiting more capillaries and is determined principally by capillary densityMuscle O.Myth #1), in healthy skeletal muscle, most capillaries support RBC flux and are, therefore, not available to be recruited. What can, and does, change from rest to contractions is the RBC flux and hematocrit within individual capillaries.89 This is crucial because O2 delivery models109 and experimental data193 support that the primary determinant of DO2 is the number of RBCs in the capillary bed, adjacent to the muscle fibers, within RBC-flowing capillaries. Accordingly, diseases such Type II diabetes92 and HF90 that decrease the proportion (and absolute number) of capillaries that support RBC flux, at least in animal models, are characterized by low DO2.194; rev.195 As discussed below in “Oxygen transport from capillary to mitochondria” the dependence of DO2 on capillary hematocrit may depend upon recently discovered aquaporin and rhesus channels in the RBC membrane.As seen above  gradient tracking. Much like a power grid, charge created at one location can power ATP production at a spatially remote site. Such a system would defend local ATP production in the face of very low PO2 and small PO2 gradients.205 In combination with oxy/deoxy-Mb removing/producing NO to regulate cytochrome C oxidase activity such an interconnected power grid would optimize ATP production in the face of a very low intramyocyte PO2  Negate the necessity for precise capillary positioning around the fibers.This schema would: (1) Abolish dependence on long intramyocyte capillary-mitochondria O206 However, some of the strongest evidence that pericytes can constrict/dilate capillaries comes from other tissues, most notably brain212; rev.213 In particular, beautiful electrophysiological studies by Zhao and colleagues211 in mouse papillary muscle demonstrate that pericytes are electrically connected via gap junctions to ventricular myocytes. Thus, they propose that, when intramyocyte [ATP] ([ATP]im) falls, KATP channel opening hyperpolarizes myocytes, capillary endothelial cells, pericytes, and vascular smooth muscle cells; decreasing pericyte and vascular smooth muscle [Ca2+] and relaxing any extant constriction. This elegant potential mechanism would be exquisitely sensitive to Q̇O2-V̇O2 mismatch elevating Q̇O2 rapidly and specifically to ventricular myocytes with the greatest needs. That said, under physiological conditions, neither cardiac nor skeletal myocytes undergo any appreciable fall in [ATP]im even across substantial—orders of magnitude—increases of metabolic demand.215 Indeed, as demonstrated by Cain and Davies,214 it is necessary to completely inhibit creatine phosphoryltransferase with FDNB , a markedly nonphysiologic condition, to decrease [ATP] substantively during muscle contractions. Such a system might be better triggered, at least in skeletal muscle, by sensitivity to the phosphorylation potential .216Reminiscent of Krogh’s search for a mechanism by which individual capillaries might control their own hemodynamics, it has been claimed that pericytes exert a “canonical” role in regulating skeletal muscle vessel diameter.2 140 to 700+mmHg) the extent of the pericyte-associated luminal constriction was extremely small  and thus, whereas it might increase individual capillary resistance, it would not be expected to prevent RBC passage. In response to whisker stimulation in mice, somatosensory cortex capillaries dilated ∼5% to 7% of their 4.4 µm baseline within ∼4 to 10 s208—faster than some arterioles and considered, by the authors, to be responsible for 84% of the regional blood flow increase. These kinetics are far faster than measured for bulk brain blood flow218 where the mean response time is ∼80 s suggesting that pericyte action may need to redistribute the existing blood flow in response to elevated regional demands. Interestingly, in brain, ischemia induces pericyte constriction of capillaries which may be irreversible.208Moreover, even under highly nonphysiological conditions  it would be necessary to demonstrate several phenomena; including: (1) That, in resting muscle, pericytes are constricting capillaries and occluding RBC flux. As evidence falsifying Myth #1 above  skeletal muscle capillary hyperemia following the onset of contractions , chemical stimuli,229 and the KATP-channel blocker glibenclamide,101 as well as the pathological derangements  appear to be mediated exclusively at the arteriolar level. Specifically, capillary hemodynamics are accelerated in hyperemic states and decelerated in low flow conditions without any discernible narrowing of the capillary lumens whether caused by pericytes or other means.229 It is also pertinent that, in resting or contracting muscle at sarcomere lengths below ∼2.7 µm , capillary RBC velocity and flux through individual capillaries exhibit enormous heterogeneity and do not correlate with capillary luminal diameter.230Perhaps, there might be a role for pericytes improving Q̇O2max were driven almost exclusively by changes in muscle(s) perfusive O2 flux  with little attention paid to muscle O2 diffusing capacity (DO2). However, some 3 decades ago Peter D. Wagner graphically  changes in V̇Ophically  conflate2 from the venous effluent, at V̇O2max when the mitochondrial were O2-supply limited, was the presence of a finite DO2.232; rev.233 As demonstrated in 2 or O2 content may be little impacted by exercise training or HF, for example, but conceal substantial changes in DO2. For instance, despite only a small increase in a-vO2 difference, in response to exercise training ∼70% of the increased V̇O2max is attributed to elevated DO2 rather than Q̇O2.232 For HF patients a reduced DO2 contributes substantially to their lowered V̇O2max even though muscle venous effluent PO2 may be very low.234; rev.195 Thus, fractional O2 extraction is synonymous with a-vO2 difference and determined by the ratio of DO2-to-Q̇which demonstrated effectively that the reason skeletal muscle could not remove all the O2 dissociation curve in the relevant physiological range.where β is the slope of the O2108,193 coheres with the capillary neogenesis stimulated by exercise training increasing, rev.236 and the capillary involution and high proportion of capillaries that do not support RBC flux in HF decreasing, 194 DO2 : Dissociate from hemoglobin, traverse the RBC membrane, plasma, capillary endothelial surface layer , endothelial cell, interstitial space, myocyte sarcolemma and intervening cytoplasm, cross the mitochondrial membranes, and react with reduced cytochrome c oxidase and protons  by phosphorescence quenching and intramyocyte PO2 (PimO2) by proton NMR and cryomicrospectrophotometry support that the bulk of the apparent resistance to O2 flux lies in close proximity to the capillary. Thus the majority of the PO2 decrease between RBC and mitochondria occurs from capillary to interstitial space and from there into the myocyte .imO2 is low 140 and absent of marked transverse or longitudinal gradients (canine gracilis).135As O protons . rev.1072’s pathway from RBC to mitochondria.Let’s consider individual steps in O2 dissociation has a 4 ms half-time with a velocity constant ∼3 000 mM/s under physiological conditions.2max, PO2 decreases <4 mmHg from Hb to the RBC membrane.240 Exciting recent data suggest that two channels  facilitate transmembrane O2 flux241 and may account for over half of the RBC O2 diffusing capacity.242 It remains to be determined how knocking out these channels impairs skeletal muscle DO2 and exercise capacity in vivo.Hemoglobin O2 sources. Thus the proportion of the capillary endothelium relevant for O2 flux is determined by how many capillaries sustain RBC flux as well as their length and crucially their hematocrit.109 Acting like an O2 bottleneck the RBC-endothelial region concentrates the O2 flux density forcing a large fall in PO2 across the microvascular-to-interstitial space.rev.119 It remains to be determined exactly how high capillary hematocrit rises during heavy or severe intensity exercise. Measurements using Time Resolved Near Infrared Spectroscopy (TRS-NIRS) in human muscles during severe intensity exercise indicate that muscle hematocrit may only increase up to 30% above resting122,2 elevation necessary for the 100-fold increase in blood-muscle O2 flux.Oxygen’s insolubility in plasma dictates that only RBCs themselves are quantitatively important OmvO2 and PisO2 there is a marked fibre type heterogeneity. Specifically, Q̇O2 at rest and during exercise is maintained far higher, with respect to V̇O2  for slow- versus fast-twitch muscles, such that their PmvO2 is higher.139 Quantifying the PO2 profile across the microvascular and interstitial compartments at rest and during contractions within contrasting fibre-type muscles helps partition the apparent O2 flux resistance into myocytes. The 1-2 µm region from the RBC membrane to the myocyte subsarcolemmal space is considered an O2 carrier-free region (CFR) encompassing the plasma and endothelial surface layers, endothelial cell, interstitial space and sarcolemma . Thus, despite the contractions-induced increase of capillary hematocrit, the plasma-glycocalyx-endothelial cell interface presents an appreciable apparent resistance to blood-myocyte O2 flux amounting to ∼60% (soleus) to 85% (white gastrocnemius) of the capillary-myocyte PO2 drop: which is consistent with Roy and Popel’s246 theoretical modeling in athletic and nonathletic animal muscles.With respect to Prcolemma . InteresmvO2 (rest and contracting) and greater PisO2 promotes a higher trans-sarcolemmal O2 flux and may serve to sustain a higher intramyocyte PO2 (PimO2). This higher PimO2 will reduce metabolic perturbations  and improve muscle contractile performance.141,rev.2 124,250In slow- versus fast-twitch muscles the higher P2 decreased proportionally with distance “R” from the capillary (Myth #4 above). Muscle regions distant from capillaries exist in an anoxic state  at rest and become oxygenated during contractions. The role of Mb in intramyocyte O2 transport was not appreciated.The theoretical model of Krogh and Erlang hypothesized that muscle POapillary  #4.21–232 microelectrode facilitated construction of tissue PO2 histograms revealing mean PO2 values between 16 and 39 mmHg in resting skeletal muscles.252; rev.253 Later Honig and colleagues’135 cryomicrospectrophotometry resolved submyocyte myoglobin O2 saturations, which permitted calculation of local intramyocyte PO2’s in rapidly frozen resting and contracting dog gracilis muscles. Despite technical shortcomings, that precluded spatial resolution much below 50 µm,254 their data showing low intramyocyte PimO2s during contractions—and therefore absence of appreciable intramyocyte PO2 gradients—was confirmed by proton-NMRS in human muscles. Specifically, in human muscles PimO2 was reduced from 20 to 30 mmHg at rest to between 2 and 5 mmHg during heavy-maximal exercise  hyperbolically1 according to:Within human muscles in particular there is an interdigitating mosaic of contrasting fibre types and a dissociation anatomically between capillary modules and muscle fibers within an individual motor unit. Accordingly, the same capillaries may supply both quiescent and active muscle fibres2 is in l/min.where V̇O2 movement from capillary-mitochondria must be consistent with the physiological observations characterized in equation 4.125 It is argued below that a contemporary understanding of O2 movement across/within myocytes is not consistent with the necessity for de novo capillary recruitment from rest-exercise nor the importance of capillary–mitochondrial diffusion distances with respect to O2 supply.Any model of capillary function and Orev.259 and also a pannexin/purinergic-dependent pathway262 and contribute to the rapid hyperemia .259 There may also be fine-tuning of capillary RBC flux across capillary modules resulting from venular vasomotor control.102 As discussed above,  evidence for unitary capillary RBC flux modulation by pericytes (Rouget cells) in skeletal muscle—at least in health and with kinetics that can account for the increased capillary RBC flux measured—is wholly absent. Moreover, the presence/participation of precapillary sphincters50; rev.158 or a contractile endothelium are not supported by either anatomical or physiological evidence.Abundant evidence demonstrates that, in skeletal muscles, the locus of capillary RBC flux control resides in the arterioles with some modulation from vasodilation signals arising from within individual capillaries.Myth #3, previously, we considered that the capillary 3D geometry, tortuosity, and branching, particularly in passively shortened or contracting muscles,128 supports the Hill model of O2 delivery. Critically, for the Hill model the dependent tissue volume decreases with distance from a given capillary and/or the myocyte sarcolemma . In addition, for horses and steers running at V̇O2max, muscles with the highest [Mb] were those calculated to have the greatest diffusion limitation .272 An extensive modeling literature also supports the importance of Mb-facilitated intramuscular O2 transport.274 275; rev.276Mb has been regarded as an O273; rev.272 but for terrestrial mammals and especially human locomotory muscles [Mb] may only be ∼0.5 mM.277; rev.276 Thus, in the extreme case of maximal exercise in human muscle (PimO2 2-3 mmHg giving ∼50% Mb desaturation) 140 this ∼6 ml O2/kg represents just <1 s of the extant V̇O2.Diving mammals may have extremely high [Mb] in their muscles; rev.266; see also253 Mb’s function as an O2 transporter is confounded by its large mass of 17,500 kDa and 3.5 nm diameter, which restricts mobility and compromises diffusion.280; rev.266 Considering its size and concentration the effective conductance of Mb-O2 is so low that it is thought to approximate that of free O2.276 Accordingly, whether Mb has a quantitatively important role in intramuscular O2 transport has been questioned.281 282; see alsofor low Mb diffusivity That said, during exercise Mb does partially O2 desaturate in contracting muscles  permitting development of an Mb-O2 gradient facilitating Mb-mediated O2 diffusion. Thus, at low metabolic rates/rest PimO2’s ∼20 to 30 mmHg143 essentially fully oxygenate Mb and would therefore extend the CFR (O2 carrier-free region) from sarcolemma into the myocyte precipitating a so-called functionally O2-carrier depleted region (FCDR).125 With Mb deoxygenating during exercise greater O2 transport capacity would be recruited as the FCDR disappears thereby increasing muscle DO2. Although speculative, this Mb mechanism (reduced FCDR) may potentially explain a portion of the rest-exercise increase of DO2 that cannot be attributed to events in the capillary .125Although championed by the Wittenbergs284 would seem to mitigate against an obligatory role for Mb. However, these animals do exhibit high in utero mortality, greater systemic hematocrit and coronary blood flow reserve and capillarity but impaired cardiac and exercise performance.286 It is not presently known whether larger species of Mb knock-outs with greater muscle fibre cross-sectional areas are viable.The fact that Mb-less mice can exercise2 transport there is evidence that diffusion distances, in-and-of themselves, might not limit O2 delivery. If this were indeed the case one raison d’etre for Mb would not exist. Specifically, Hudlicka and colleagues287 noted that muscles with contrasting intercapillary distances exhibited very similar muscle/mitochondria-specific metabolic rates.see also288 289 Also, DO2 at V̇O2max in dog gastrocnemius-plantaris is not related to capillary density and therefore mean intramuscular diffusion distances after immobilization or exercise training.196 Unfortunately, those studies could not determine the RBC volumes within the capillary bed, and changes thereof, that constitute a primary determinant of DO2.109Notwithstanding the above support for Mb’s importance in facilitating intramyocyte O2 diffusion  Gros et al.276 have calculated that, given the root mean square displacement for Mb-O2 between ∼4 to 12 µm, O2 must combine and dissociate multiple times to traverse the necessary intramyocyte distances . Moreover, Mb-O2 must cross various potential barriers such as sarcoplasmic reticulum, myofilaments and T-Tubules although M-Lines and Z-disks may not impede this process.290; rev.107 It is also pertinent that, whilst the low intramyocyte PO2’s143 might facilitate O2 transport via Mb, in part by removal of the FCDR, absence of substantial intramyocyte PO2 gradients, from subsarcolemmal to more capillary-distant cell regions, would presumably decrease the quantitative importance of passive relative to Mb-facilitated O2 diffusion.In addition to this circumstantial evidence questioning the central role for Mb-facilitated O254,142 analyses, though spatially crude, support that intramyocyte PO2’s remain >1 mmHg. This value is far greater than the “critical” PO2, below which V̇O2 is constrained .294 In contrast, mitochondrial V̇O2max versus intramyocyte PO2 curves generated by humans inspiring a range of hypoxic, normoxic, and hyperoxic gases  support a far higher critical PO2 ∼4 mmHg; as do measurements made by phosphorescence quenching in the rat spinotrapezius muscle.296 Interestingly, spectroscopic determination of muscle [oxidized cytochrome C oxidase ] at V̇O2max reached a nadir common with that for death or anoxia,297 which were interpreted as evidence for an ∼2 mmHg cytosol-mitochondrial PO2 gradient.272 However, with an estimated mitochondrial surface area being >100 times that of the functional capillary  the corresponding O2 flux density would be tiny; requiring a disappearingly small transmitochondrial membrane(s) PO2 gradient.299Both cryomicrospectrophotometrici.e., see in272 su2 transport function of Mb the novel concept has been advanced that, depending upon local O2 availability, Mb switches between NO scavenging and NO production. Thus, at higher PO2’s, extant for example in resting muscle, Mb continuously produces nitrate (NO3−) from NO raising intramyocyte [NO3−] above plasma concentrations . When intramyocye PO2 falls precipitously during intense exercise  from which free NO dissociates, triggering NO-mediated suppression of oxidative function.+], [xanthine], and [hypoxanthine]305 transposes the energetic burden from intermyofibrillar to subsarcolemmal mitochondria thereby preserving intramyocyte O2 flux and forming what Clanton205 has insightfully termed the “NO shield”  gradients to move spatially like a power grid. In this manner charge generated at one location can produce ATP remotely. The elegance of this system provides a robust protection against focal hypoxia limiting ATP generation and abrogates dependence on long O2 diffusion pathways from capillary to mitochondria. Without the opportunities for the “lethal corners” of tissue anoxia hypothesized by Krogh, this schema helps explain the dissociation between capillary density (therefore diffusion distances) and DO2 noted by Hepple and colleagues.196; see alsoIn contrast to the classical depiction of mitochondria as independent “bean-shaped” organelles, in myocytes, of all mammalian fibre types, they constitute a contiguous interconnected reticulum see  that may2 can correlate with capillary volume density307 As above, this observation is consistent with DO2 being determined principally by the volume of RBCs adjacent to the contracting muscle fibers109; see also193 and mandates using 3D models with RBC volume fractions to calculate muscle oxygenation.308 The DO2 dependency on capillary RBC volume means that RBC distribution and hemodynamics can, via altered DO2, impact PimO2 regulating muscle metabolic control and therefore contractile performance.Among species and muscle fibre types and across exercise training programs muscle DO2 it is important to consider all the potential pathways of intramyocyte O2 transport  above that provided by the direct capillary–sarcolemmal “contact” considered by Ellis and colleagues when proposing the Hill model.130In addition to upstream  determinants of DOransport . We have2 transport is very different from that hypothesized in the Krogh model. Tissue and mitochondrial volume decrease with increasing distance from the sarcolemma  capillaries in resting muscle do not support RBC flow (see left side of In contrast to the right side of mvO2) via reduced driving pressure and also a sympathetically induced arteriolar vasoconstriction.311Anesthesia, especially in combination with systemic hypoxia, can cause hypotension decreasing muscle Q̇  in resting and exercising muscles across the fibre-type spectrum  and muscle dilation (hypoxia). The former will have direct consequences for decreasing the proportion of capillaries that support RBC flux. Phosphorescence quenching measurements have defined the microvascular and interstitial POtrum see  #4131–13Sharp or blunt force trauma during surgery can prevent arterioles and capillaries flowing. Aggressive removal of overlying fascia to improve visual clarity may also damage the microcirculation and should be avoided/minimal.88 Stretching muscles also produces a reflex vasoconstriction, which compounds the hyporemia and capillary no-flow.312Muscle stretch above 3 µm, often practiced to thin the muscle and improve microcirculation optical clarity, stretches the capillaries, reducing their luminal diameter and vascular conductance and stopping RBC flux in a substantial proportion of capillaries.2 (PimO2), evoked consequent to altered PmvO2 and PisO2 (and thus PimO2) can exert a commanding influence on myocyte energetics, substrate utilization and metabolic control.; rev.313; see also314 Moreover, inattention to establishing physiologic PO2 profiles can destroy our ability to, for example, determine the effect of manipulating the nitrate-nitrite-NO pathway on contractile performance.315Attention to these and other physiological conditions  insofar as possible, is simply good science. Where such a standard is not met, the results must be interpreted accordingly. With respect to muscle oxygenation this is especially true, not just for microcirculatory, but also for metabolic control. Specifically, even disappearingly small changes in intramyocyte POEffective progress in muscle microcirculatory research will be facilitated by:Advocating for rigorous quality control of intravital microscopy investigations and interpreting previous findings, theories, and models accordingly.Recognizing that different organ microcirculations subserve wide-ranging functions and those elements critical for capillary hemodynamic control in muscle may work very differently for heart, mesentery, gut, and especially the brain.208,210,316 and heart,211 with skeletal muscle89). Is there really the possibility for unitary capillary control in muscle, or rather, is fine-tuning of Q̇O2-V̇O2 matching achieved by ascending vasodilation that increases terminal arteriole diameter and thus RBC flux to all capillaries in a given capillary module.106,106Testing novel or extant hypotheses within the context of physiological control. For instance, as regards the role of pericytes in closing/opening capillaries, ask whether their response kinetics are sufficiently fast to support the Q̇ and capillary hemodynamics demonstrated following the onset of contractions? In brain and heart the pericyte-associated constriction is slight and too slow to explain the hyperemia kinetics following contractions onset  there is evidence during knee-extension exercise that estimated capillary Q̇O2 kinetics may be slower  than that of the femoral artery and this phenomenon is expected to produce a deoxygenation overshoot. Future studies might judiciously account for capillary hemodynamics rather than relying on the tacit presumption that such kinetics are the same as those for arterial Q̇.The elegant NIRS technology employed by Bowen and colleagues2. Rather, a key determinant of DO2 is the RBC volume of flowing capillaries, which may reflect the importance of newly discovered aquaporin and rhesus pores in the RBC membrane and also the high RBC-capillary endothelium O2 flux density.Recognizing that strong physiological evidence supports that capillary–mitochondrial diffusion distances do not determine DO243Understanding more about the role of the endothelial surface layer  in determining capillary hematocrit and hemodynamics, especially during exercise, in health and perturbations of such in disease. In vivo NIRS measurements during heavy intensity exercise suggest that capillary hematocrit may not reach systemic levels even at very high muscle Q̇s.2 can diffuse between arterioles, capillaries, and venules266 and the presence of higher PisO2 than (independently) measured for PimO2 raises the intriguing possibility that O2 movement through the interstitial space—around the myocyte periphery and among myocytes—may be important. If so, this provides additional support for the Hill (versus Kroghian “pinpoint”) model of O2 diffusion  in one region might be beneficial if total muscle Q̇O2 is constant and such behavior therefore predicates a low PmvO2 in another region.Considering that Oiffusion 107,130 a2 transport—aqueous and myoglobin-facilitated O2 diffusion and, potentially, that O2 tracks along lipid membranes during heavy and severe exercise (2 sensitive dyes with an active range ∼0.5 to 10 mmHg and rapid response in the subsecond range will be invaluable to this mission.Determining the quantitative importance of the multiple pathways for intramyocyte Oexercise . This isexercise . DevelopIncorporating the latest aspects of muscle capillary function models e.g.,  into the


Therefore, SOC is not explicitly included in the current model estimation of the global forest CH4 sink. Here, using in-situ observations, global meta-analysis, and process-based modeling, we provide evidence that SOC constitutes an important variable that governs the forest CH4 sink. We find that a CH4 sink is enhanced with increasing SOC content on regional and global scales. The revised model with SOC function better reproduces the field observation and estimates a 39% larger global forest CH4 sink (24.27 Tg CH4 yr−1) than the model without considering SOC effects (17.46 Tg CH4 yr−1). This study highlights the role of SOC in the forest CH4 sink, which shall be factored into future global CH4 budget quantification.Soil organic carbon (SOC) is a primary regulator of the forest–climate feedback. However, its indicative capability for the soil CH Soil organic carbon has a positive effect on the removal of methane in forest soils. Global forests are found to be larger sinks of methane than previously estimated when the influence of SOC is considered. Among upland ecosystems, forests play a pivotal role, accounting for up to 62% of the global soil CH4 sink3. Therefore, accurate quantification of the global CH4 sink provided by forest ecosystems is essential to improving our understanding of the global CH4 budget.Upland soils are an important sink of atmospheric methane  as major determinants of soil CH4 sink8. Along with soil gas diffusivity, other soil variables such as alternative carbon sources10 and nutrient availability12 have been suggested to play a pivotal role in determining the soil CH4 sink. Incorporation of physiological characteristics of methanotrophs into the process-based model indeed constrained the uncertainty of estimation14, suggesting that it is crucial to incorporate them into the model to improve our estimation capability. However, due to our incomplete understanding of the regulatory mechanisms behind these variables, current estimations do not adequately consider these variables in the model14. Thus, investigation of the regulatory mechanisms of the previously overlooked drivers and further consideration of these factors is needed to reduce the uncertainty of global forest CH4 sink estimation.The magnitude of global forest CH17. While SOM data has traditionally been used as a soil quality indicator in soil science, various disciplines are now acknowledging the significance of SOM attributes as indicators of ecosystem processes, including climate feedback. Therefore, diverse ecosystem process models now incorporate quantitative and qualitative data on SOM18. Previous models generally considered SOM to negatively affect the soil CH4 sink because it can provide carbon substrates to methanogens, thereby increasing CH4 emissions19. However, this assumption does not consider the diverse mechanisms of SOM-dependent regulation of soil variables that are associated with soil CH4 oxidation, constraining our estimation capability.Soil organic matter (SOM) is a complex of decaying organic matter, degradation products such as humus, and soil microbes, and plays a pivotal role in terrestrial ecosystem functioning4 sink and to further improve global forest CH4 sink estimation, three independent approaches—continuous in-situ measurement, global meta-analysis, and process-based modeling—were used in this study. First, we monitored in-situ CH4 uptake rate and edaphic characteristics, including SOM content, for 2 years in subtropical forests with different tree species  and in temperate coniferous forests with different thinning intensities . Second, we performed a global meta-analysis to establish a relationship between the forest CH4 sink and soil organic carbon (SOC) content on a global scale. Finally, we revised a process-based forest CH4 sink model by considering the effect of SOC content to quantify the global forest CH4 sink.To elucidate the mechanisms underlying the SOM-dependent control of the forest CH4 fluxes and edaphic characteristics for two years from April 2018 to March 2020 revealed there are significant positive correlations between SOM content and CH4 uptake rate both in a subtropical forest with different tree species  and in a temperate coniferous forest with different thinning intensities . The positive relationship observed in temperate forest further suggests that the variability of CH4 uptake between the forests with the same dominant species but different soil characteristics can be partially explained by the SOM content. It is conceivable that the positive correlations observed between SOM content and CH4 uptake rate at the two examined sites may have been primarily influenced by other environmental variables that exhibit variability according to tree species or thinning intensity and that are closely associated with CH4 uptake. However, partial correlation analysis indicates that even after removing the influence of other variables such as air-filled porosity (AFP), dissolved organic carbon (DOC) content, and water-filled pore space, SOM content remains positively correlated with CH4 uptake rate  has a unimodal relationship with soil CH4 uptake rate30. Under low soil moisture, water stress can promote ethylene production by plants, which inhibits CH4 oxidation in soil31. In addition, water stress on methanotrophs can result in reducing their activity29. On the other hand, high soil moisture inhibits methanotrophic activity by decreasing gas diffusivity30. A global meta-analysis showed that the forest CH4 sink increased with increasing mean annual precipitation, suggesting a higher CH4 uptake at sites with more precipitation on a global scale32. These authors suggested that the positive effect of water content induced by water-stress alleviation is larger than the negative effect of decreased CH4 and O2 availability in the forests. Several other studies indicated the prevalent role of precipitation in promoting biological oxidation of CH4 in forest soils34, suggesting that enhanced water-holding capacity may stimulate methanotrophic activity, with the exception of highly saturated periods. Although the correlation between SWC and CH4 uptake rate was not significant at our study sites, the highest CH4 uptake rate was observed in the oak forest , and temperate forest , indicating that soil moisture positively influences soil CH4 uptake, with the exception of periods of high soil water saturation. Significant positive correlations between SOM content and SWC both in the subtropical forest and temperate forest , suggesting that SOC indeed positively influences forest CH4 uptake in global forests. In addition, by comparing four ranges of SOC content, we confirmed that CH4 uptake rate increases with the increasing SOC content  when SOC content ranged from 60 to 100 g kg−1. While SOC has been regarded to rather reduce the forest CH4 sink by affecting methanogenesis32, the relationship we found in the field observations and meta-analysis suggest that the positive effect of SOC on soil CH4 uptake outweighs the negative effect at both regional and global scales. These results further suggest the need to incorporate the positive effect of SOC into the estimation model.To provide the global context for our in-situ measurements, we retrieved data on mean forest CHles Fig. . A signient Fig. : mean so4 sink. Overall, a comparison between the observed and simulated forest CH4 uptake rates showed a 30% reduction of root mean square error (RMSE) values in the new model that included the function of SOC . However, when different biomes were analyzed separately, estimation using the new model substantially reduced RMSE in temperate and boreal forests but increased it in the tropical forest. This result indicates that the simulated CH4 uptake rate better represents the observational data in temperate and boreal forests by considering the effect of SOC, but not in the tropical forest.We incorporated the effect of SOC into the terrestrial ecosystem model (TEM) to quantify the global forest CH4 sink as 24.27 ± 1.64 Tg CH4 yr−1, which is 39% larger than the estimate by TEM-DEF (17.46 ± 5.09 Tg CH4 yr−1)  and boreal forest (5.82 ± 1.27 Tg CH4 yr−1 versus 1.32 ± 0.44 Tg CH4 yr−1), respectively, whereas estimated 23% lower CH4 sink in tropical forest (4.65 ± 0.37 Tg CH4 yr−1 versus 6.06 ± 2.8 Tg CH4 yr−1) , followed by middle latitudes (20–40° N&S), and shallowest at low latitudes (0–20° N&S)  Fig. . The dif4 sink on both regional and global scales. Our revised model with SOC function improves the estimation and reconciles the disparity between bottom-up estimation and top-down modeling. Revealing the response of climate feedback to changing SOC dynamics is one of the major challenges for projecting future climate change. SOC stocks in terrestrial ecosystems including forests are sensitive to climate change and are projected to be reduced by global warming51, which may induce a positive feedback to climate change. The global soil CH4 sink is projected to increase in the future, most likely due to the increase in atmospheric CH4 concentration and temperature rise52. However, our study suggests that the reduction of the SOC stock in forest ecosystems by warming can partially offset the increasing soil CH4 sink, potentially inducing positive feedback to warming along with the increasing CO2 emission. Thus, it is important to incorporate the effect of SOC on the forest CH4 sink to better understand the impact of climate change and constrain the global CH4 budget.Our study based on field measurements and global meta-analysis provides evidence on the pivotal role of SOC in the forest soil CHQuercus acuta (oak), Cryptomeria japonica (Japanese cedar), and mixed forest. In temperate forest, we selected Pinus koraiensis dominant forest with 3 different thinning intensities: light intensity (700 trees ha–1), moderate intensity (600 trees ha–1), heavy intensity (500 trees ha–1), and control (800 trees ha–1). Soil samples were collected seasonally  in 2018 and 2019 at subtropical forest and monthly from April 2018 to March 2020 at temperate forest. Soil samples were collected to the depth of 0–10 cm after litter layer and O-horizon removal. Soil temperature was measured at the depth of 5 cm after soil sampling. Collected samples were placed in plastic zipper bags and transported to the lab on ice. Samples were stored at 4 °C and further analyses were conducted within 3 days.The study sites were located in the Hannam experimental forest  and in Gwangneung experimental forest  in the Republic of Korea. The mean annual temperature was 17.0 °C and 11.7 °C in subtropical forest and temperate forest, respectively, and the mean annual precipitation was 2188 mm and 1364 mm during the observation period in subtropical forest and temperate forest, respectively. In subtropical forest, we selected 3 different tree species: CHP, Shimadzu, Kyoto, Japan). Soil core of 0–10 cm depth was collected to measure bulk density. Based on the measured bulk density, Air-filled porosity was calculated as below:s is particle density (2.65 g cm–3) and θv is volumetric moisture content.SWC was measured by oven-drying at 105 °C for 24 h, and SOM content was determined by a loss-on-ignition method. Soil DOC content was extracted by mixing soil with distilled water, filtered through 0.45-µm filter, and then analyzed by a TOC analyzer  from April 2018 to September 2019. Due to mechanical issues with the portable analyzer, we took the headspace gas sample every 10 min for 50 min and transferred it to a pre-evacuated glass vial from October 2019 to April 2020. We used a GC-FID  to measure the CH4 concentration. CH4 fluxes were calculated from linear regression slopes (chamber headspace [CH4] vs. time) with a minimum R2 = 0.82.Soil CH4 uptake OR CH4 sink) AND (forest)’. Reported variables including annual CH4 uptake rate, topsoil SOC or SOM content, mean annual precipitation, mean annual temperature, latitude and longitude were extracted from each paper. Collected SOM contents (N = 5) were converted to SOC content by using the simple equation: SOM = SOC × 1.72 (van Bemmelen factor53). The van Bemmelen factor has been widely used to convert SOM content to SOC content54.We also collected papers that present only total C content (N = 26) since inorganic C content in forest soil is negligible56. When the paper presented SOC values with depth profiles, values below the humus layer were collected. In the case of field manipulation experiments such as warming, elevated CO2, and fertilizer addition, the CH4 uptake rate of only the control plot was collected to prevent other controlling variables affecting the CH4 uptake rate of forests with natural conditions.We collected 204 data points from 81 published papers using Web of Science and Elsevier’s Scopus academic database. We searched articles published between 1989 to 2021 using the keyword ‘ and limiting functions of methane concentration, soil temperature, soil moisture, redox potential, nitrogen deposition, and diffusion limited by high soil moisture  (Eq. 4 oxidation function by incorporating the SOC effect on CH4 oxidation (f(SOC) in Eq. 4 oxidation using our collected data  Eq. . For the4 oxidation related to both TEM-DEF and TEM-SOC for tropical, temperate, and boreal forest regions (Supplementary Table 8. We used the Shuffled Complex Evolution Approach in R language (SCE-UA-R) to minimize the difference between simulated and observed CH4 oxidation rates58.We optimized five parameters related to maximum oxidation potential, soil temperature, and moisture sensitivity for CH4 emission and consumption, we used spatially explicit data of land cover, soil pH and textures, meteorology, and leaf area index. We used SOC data from Soilgrids database59. The model was applied at the spatial resolution of 0.5° latitude by 0.5° longitude for forest upland ecosystems with a daily time-step during 2000–2016. A year of spin-up was used for CH4 equilibrium in soils, and simulated ecosystem-specific CH4 oxidations were then area weighted for each grid cell. For each site, 20 ensembles were run using SCE-UA-R with 10,000 maximum loops per parameter ensemble, and all of them reached steady state before the end of the loops.To make spatially- and temporally-varying estimates of CH4 uptake rate in the field. In the model, tree species  or thinning intensity (temperate forest) was used as a random effect for the incorporation of between-species or between-thinning intensity dissimilarity. Before conducting regression analysis, SOM content and soil CH4 uptake rate was log-transformed to improve the normality of the data and validity of regression analysis. Correlations between soil characteristics  and SOM content as well as soil characteristics and CH4 uptake rate were assessed with Pearson’s product moment correlation coefficients. Linear mixed effect model was used to assess the effect of tree species or thinning intensity on forest soil CH4 uptake rate, SOM content, soil AFPS, and SWC. Tree species or thinning intensity was used as categorical variables, and plot and month were used as random effects. Tukey’s HSD tests were used to compare individual means. Partial correlation analysis was conducted to assess whether SOM content was positively correlated with soil CH4 uptake rate without the influences of other variables that vary by tree species or thinning intensity. In this analysis, environmental variables that (1) vary by tree species or thinning intensity and (2) are closely related to soil CH4 uptake rate were selected as control variables.Linear mixed-effect models were used to assess the positive relationship between SOM content and soil CH4 uptake rate also exists in the global forests. Before conducting linear regression analysis, collected soil CH4 uptake rate and SOC content were log-transformed to improve the normality of the data and the validity of the analysis. To assess whether forest soil CH4 uptake increases along the SOC content range, we conducted Kruskal–Wallis one-way ANOVA.Using the data we collected from 81 previously published papers, linear regression analysis was conducted to test whether positive relationship between SOC content and soil CHFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Our results on steady-flow and oscillatory measurements indicated that shear thinning properties can be fitted well by the Herschel–Bulkley model and by the formation of highly interconnected gel structures in the complexes, respectively. Analyzing the rheological and structural features simultaneously led to an understanding that formations of extra junctions and the rearrangement of the particles in the CSM–Blg–Ca could enhance elasticity and viscosity, as compared with the effect of CSM–Blg complex without salts. NaCl reduced the viscosity and dynamic rheological properties and intrinsic viscosity through the salt screening effect and dissociation of structure. Moreover, the compatibility and homogeneity of complexes were approved by dynamic rheometry based on the Cole–Cole plot supported by intrinsic viscosity and molecular parameters such as stiffness. The results outlined the importance of rheological properties as criteria for investigations that determine the strength of interaction while facilitating the fabrication of new structures in salt-containing foods that incorporate protein–polysaccharide complexes.Functional, physicochemical, and rheological properties of protein–polysaccharide complexes are remarkably under the influence of the quality of solvent or cosolute in a food system. Here, a comprehensive description of the rheological properties and microstructural peculiarities of cress seed mucilage (CSM)-β-lactoglobulin (Blg) complexes are discussed in the presence of CaCl Liquid–liquid phase separation or complex coacervation involves interactions between molecules that ultimately make charged polymers bind with each other, especially in protein–polysaccharide interactions . Where cWith its small size, β-lactoglobulin (Blg) is a compact, dominant, globular part of whey protein, and has received considerable attention due to its extensive functional properties  . Due to Lepidium sativum. The molecular weight of CSM is approximately 540 kDa. Its zeta potential is −10.78 ± 0.19 mV and it is characterized by an intrinsic viscosity of 13.3 dL/g. While it is regarded as a polyelectrolyte molecule, CSM comprises a rigid chain conformation and contains D-galacturonic acid and D-glucuronic acid  between Blg and CSM with cosolute (NaCl and CaCl2) [+ ion, an increase in I from 0.01 to 0.1 M resulted in a drop in CSM–Blg intrinsic viscosity compared to Ca+2 from (0.46 to 0.18 dL/g) and (0.46 to 0.28 dL/g), respectively. Such a decrease with the addition of NaCl can be assigned to the enhanced charge screening of polymer chain inducing more contracted conformation and causing a reduction in the hydrodynamic size of the molecule [2 in dispersions, intrinsic viscosities decreased (from 0.46 to 0.32 dL/g and from 0.46 to 0.25 dL/g) in the presence of 2 and 5 mM CaCl2 (The intrinsic viscosity (r chains . The resosolutes . As indiosolutes . This cod CaCl2) . In the molecule . With thmM CaCl2 , respect2 showed that most of the particles are clustered together, forming a rod shape. These big microparticles are mainly composed of small nanoparticles in various size ranges . 2 which exhibited a certain change in morphology with an organized structure. Some particles are tightly combined, and nanoparticles and microparticles showed a tendency to form an organized structure. i.e., hexagonal-like. In other words, the rearrangement of the particles to new structures could result in the variation of the rheology of this particle at high concentrations.For a deeper insight into the effect of the high concentration of calcium, SEM image analysis was carried out see . SEM ima−1, 1637–1644.5 cm−1, 1644.5–1662 cm−1, 1662.5–1682 cm−1, and 1682–1689 cm−1, respectively. These parameters were previously confirmed by [−1 for Blg. This peak describes how the native protein is organized in solution by the β-sheet. After the formation of the nanocomplex, a shift can be observed to 1630 cm−1 and 1628 cm−1 in Blg–CSM and Blg–CSM–Ca3, respectively, highlighting the possibility that Blg preserved its structures, suggesting the role of CSM in the reorganization of structure. A relatively big surface area exists in the β-sheet structure and can benefit the formation of ordered hydrogen bonds [FTIR spectroscopy is a sensitive diagnostic tool for monitoring conformational alterations in biopolymers. Considering Blg, the observed peaks correspond to the β-sheet, random coil, α-helix, β-turn, and β-antiparallel sheet at 1613–1637 cmirmed by . In our en bonds . Thus, ien bonds .2+) had a more prominent effect on intrinsic viscosity of CSM–Blg, compared to the monovalent type (Na+). A possible reason for this is the occurrence of molecular crosslinking among CSM and calcium ions, as well as various degrees of aggregation which lead to a diverse extent of molecular contraction [coilR) and its corresponding volume (coilV) in CSM–Blg complexes was strongly affected by ion type and concentration, as demonstrated in coilR values of CSM–Blg complexes were higher and were reduced with an increase in the salt level, which could be ascribed to less-extended CSM chains and more compact structure. The changes of intrinsic viscosity, coil radius, and volume are interdependent. These findings are corroborated by changes in the intrinsic viscosity of CSM–Blg samples. As an indicator of polymer chain stiffness, salt tolerance correlates with a higher degree of chain flexibility. A higher level of dependency on ionic strength in intrinsic viscosity is usually accompanied by a greater salt tolerance. Based on Equation (17), the plot of the intrinsic viscosity was obtained vs. the reciprocal of the square root of ionic strength (I−0.5) for evaluation of the CSM–Blg stiffness parameter in the presence of Na+ and Ca2+ ions. As seen, the stiffness parameter was lower in the presence of the monovalent ion NaCl (3.34) as compared to the divalent one (CaCl2) (14.46), demonstrating a stiffer backbone of CSM–Blg in the presence of divalent ions. In other words, contraction caused by Ca2+ in CSM–Blg complex was greater than that of Na+, which is consistent with the results of viscosity and SEM images.In sum, wherever the same ionic strength applied, the divalent ion , Blg (with 93% purity), sodium hydroxide (NaOH), and hydrochloric acid (HCl) were sourced from Sigma-Aldrich . Deionized water was used for diluting the solutions when necessary .Cress seeds were locally obtained from a reputable seed seller. CaClExtraction and purification processes of cress seed mucilage were carried out appropriately . Briefly2 and NaCl were added to the aforementioned mixture, and the resultant dispersion was mildly stirred for 2 h. The final contents of CaCl2 and NaCl were in the range of 2–10 mM and 10–100 mM, respectively. Salt concentrations were set out according to the stability of CSM in the said range of salt content [To prepare stock solutions of Blg and CSM, the two components were dissolved in deionized water to reach 20 mg/mL and 30 mg/mL, respectively. Then, by post-blending acidification, CSM–Blg complexes were created. CSM was added to Blg dropwise. The variables were selected according to previous methods . For the content . Ultimatn and pk describe the flow behavior index (dimensionless) and power-law consistency coefficient (Pa·sn), respectively.Hn describes flow behavior index (dimensionless), Hk defines the consistency coefficient (Pa·sn), and τH0 is the yield stress (Pa) according to the Herschel–Bulkley model.τB0 and Bη are the Bingham yield stress (Pa) and Bingham plastic viscosity (Pa s), respectively.τC0 and Cη are the Casson yield stress (Pa) and Casson plastic viscosity (Pa s), respectively.Within 0.1–100 1/s, the various properties of steady shear flow (CSM–Blg) (20 °C) were determined by a rheometer . After applying shear thinning models, the experimental curves that describe the flow behavior were fitted by the Ostwald–de Waele (or power law), Herschel–Bulkley model, Bingham, and Casson (Equations (1)–(4)):2).The flow behavior of nanocomplex solutions was depicted by a best-fitting model which had the highest coefficient of determination (Rw/v) at three levels of concentration for NaCl  and CaCl2  via a rheometer . Before measuring the dynamic shear, each sample was maintained for 10 min in storage at 25 °C. The linear–viscoelastic region (LVR) was estimated by strain sweep determinations before frequency sweep tests, where G″ and G′ performed without dependency on strain amplitude. In this context, within 0.01–100% of strain, a constant frequency of 1.0 Hz enabled the strain sweep determination (25 °C). While the LVR circumstances applied, frequency sweep tests ran at a constant strain amplitude (0.1%). In the LVR region, 0.01–10 Hz frequencies were considered for frequency sweep determinations. Frequency dependence of the storage modulus (G′) and loss modulus (G″) was examined accordingly through the power law model [k′ and k″ (Pa·sn) are constants; n′ and n″ are the viscoelastic components of elastic and viscous moduli, and ω denotes the oscillatory frequency (Hz).The CSM–Blg had its rheological behavior of dynamic shear properties determined through a finite level of total biopolymer concentration (2% aw model :(5)G′=k′w/w total concentration) at different salt levels was investigated by Cole–Cole plots. To this end, the imaginary (η") part of the viscosity was plotted against its real (η′) component [The miscibility of different semidilute CSM–Blg complexes (2% omponent .(7)η″=Grelη) was measured along with specific viscosity (spη) as follows (Equations (9) and (10)):t and t0 are the flow times of sample and pure solvent (deionized water), respectively. The intrinsic viscosity (η) was calculated by the models below [K′ and K″ represent the Huggins and Kraemer constants, respectively.A thermostatic water bath was used for hosting a U-tube capillary viscometer  in suspension form. The conditions were under an accurate temperature control (25 ± 0.1 °C), by which relative viscosity (ls below :(11)HuggcoilR) and hydrodynamic coil volume (CoilV) were estimated by the following equations (Equations (15) and (16)) [AN is Avogadro’s number and WM (kDa) denotes weight–average molecular weight.Hydrodynamic coil radius (nd (16)) :(15)RcoiS  represents a comparative criterion for evaluation stiffness macromolecular chains [The ionic strength dependence of the intrinsic viscosity of polyelectrolytes usually follows the empirical Smidsrød and Haug (1971) equation which allows for describing the macromolecular chain stiffness depending on molecular weight:r chains .−5–10−6 Pa) was used for taking the images (5–10 kV). The structural changes were also examined by scanning electron microscopy . A drop of the suspension was dripped onto glass stubs and placed on the surface of the sample stub, which was then left to dry. The stub was then coated with a thin (<20 nm) conductive gold layer using a sputter coater  before imaging. After preparation, the morphological features of each sample were visualized by electron microscopy.To arrive at a sufficient description of variations in nanocomplexes within the specifications of salts and their features, the procedure involved placing samples on an FE-SEM stub for field-emission scanning electron microscopy. After coating each sample with an Au–Pt layer, electron conductivity was supposedly exposed to the surface of the sample. High-pressure vacuum  at 400–4000 cmp < 0.05) via SPSS 23.0 .In triplicate, the measurements were deemed reliable and with few errors. Mean values ± standard deviation were considered to present experimental data. The Origin Pro 9.1 software  was used for generating figures. Differences between means were determined by one-way analysis of variance (ANOVA). Duncan’s comparison validated the analysis of variance (


Pancreatic ductal adenocarcinoma (PDAC) has a high fatality rate, mainly due to its asymptomatic nature until late-stage disease and therefore delayed diagnosis that leads to a lack of timely treatment intervention. Consequently, there is a significant need for better methods to screen populations that are at high risk of developing PDAC. Such advances would result in earlier diagnosis, more treatment options, and ultimately better outcomes for patients. Several recent studies have applied the concept of liquid biopsy, which is the sampling of a biofluid (such as blood plasma) for the presence of disease biomarkers, to develop screening approaches for PDAC; several of these studies have focused on analysis of extracellular vesicles (EVs) and their cargoes. While these studies have identified many potential biomarkers for PDAC that are present within EVs, their application to clinical practice is hindered by the lack of a robust, reproducible method for EV isolation and analysis that is amenable to a clinical setting. Our previous research has shown that the Vn96 synthetic peptide is indeed a robust and reproducible method for EV isolation that has the potential to be used in a clinical setting. We have therefore chosen to investigate the utility of the Vn96 synthetic peptide for this isolation of EVs from human plasma and the subsequent detection of small RNA biomarkers of PDAC by Next-generation sequencing (NGS) analysis. We find that analysis of small RNA from Vn96-isolated EVs permits the discrimination of PDAC patients from non-affected individuals. Moreover, analyses of all small RNA species, miRNAs, and lncRNA fragments are most effective at segregating PDAC patients from non-affected individuals. Several of the identified small RNA biomarkers have been previously associated with and/or characterized in PDAC, indicating the validity of our findings, whereas other identified small RNA biomarkers may have novel roles in PDAC or cancer in general. Overall, our results provide a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach that relies on Vn96-mediated isolation of EVs from plasma. Survival from PDAC is significantly higher the earlier the disease is diagnosed, at a time when complete surgical removal of the tumour offers the best chance at curative treatment2. Therefore, a key challenge for PDAC is the development of a reliable assay that will detect the disease at its earliest stages. Due to the asymptomatic nature of early-stage PDAC, populations that are at high-risk of developing this disease would derive significant benefit from preventative or diagnostic screening to identify its presence. Such populations include patients that have a family history of PDAC, suffer from chronic pancreatitis, or have type II diabetes3.Pancreatic ductal adenocarcinoma (PDAC) has a very high mortality rate due to the significant number of patients (~ 80%) that have non-localized metastatic disease at the time of diagnosis4, represents a promising approach for PDAC screening and detection. Typically, nucleic acids or proteins that circulate in a biofluid are analyzed during liquid biopsy; however, isolation of extracellular vesicles (EVs) from a biofluid and characterization of their cargoes, which include nucleic acids and proteins, is gaining interest4. Recent studies have explored the use of EV cargoes as biomarkers for PDAC15. While such studies have identified differential expression of nucleic acids and proteins in the EVs of PDAC patients, the robust application of these novel biomarkers in a clinical setting requires reliable techniques for the isolation of EVs in a high-purity homogeneous form, and these methods must be amenable for use in a clinical diagnostic laboratory16.Liquid biopsy, which is the interrogation of biofluids  for the presence of disease biomarkers24. As such, the use of Vn96 enables multi-omic analysis of EV cargos including DNA, RNA and protein23. Small RNA sequencing of nucleic acid extracted from Vn96-captured EVs demonstrates an enrichment of EV small RNA and a similar sequencing profile to other commercially available EV isolation methods (ExoRNeasy), as well as the most commonly-used laboratory technique for EV capture (ultracentrifugation25). In a pre-clinical setting, Vn96 has been used to isolate and detect relevant disease biomarkers from plasma for amyotrophic lateral sclerosis26 and lung cancer24, and to isolate and detect biomarkers from urine for prostate cancer18. Together, these studies indicate that the Vn96 synthetic peptide is a promising candidate for an EV isolation method that is robust, reproducible, and applicable for a clinical diagnostics laboratory.It has been well-established that the Vn96 synthetic peptide facilitates isolation of EVs from a variety of biofluids using a simple protocol that relies on standard laboratory equipmentIn the present study we used the Vn96 synthetic peptide to isolate EVs from plasma samples obtained from PDAC patients and non-affected individuals, followed by isolation of RNA and next-generation sequencing (NGS) analyses for small RNA species. We identified differentially-expressed small RNA species in EVs isolated from PDAC patients, with the most profound differences in the expression profiles of miRNAs and lncRNA fragments between PDAC patients and non-affected individuals. Moreover, our analyses identified small RNAs that were previously found to be associated with PDAC. Our results indicate that EVs isolated with the Vn96 synthetic peptide are able to discriminate between PDAC patients and non-affected individuals through analysis of their small RNA cargoes, providing a basis for a clinically-amendable detection and/or screening strategy for PDAC using a liquid biopsy approach.We set out to study the differential expression of small RNAs, including miRNA, tRNA, and piRNA, found in EVs isolated with the Vn96 synthetic peptide from the plasma of patients with pancreatic ductal adenocarcinoma (PDAC) and non-affected ‘healthy’ control individuals. PDAC patients’ and non-affected individuals’ characteristics are shown in Table 22. Total RNA was extracted from the isolated EVs and RNA profiles for non-affected individuals and PDAC patients are shown in Fig. In total, plasma samples from 13 non-affected individuals and 16 PDAC patients were subjected to EV isolation using a previously-established protocol that relies on the Vn96 synthetic peptide27, which aligns sequencing data to the genome to first provide chromosomal regions, then annotates these regions using available databases. This type of approach has previously been used for RNA sequencing data derived from cellular RNA29, and our validation of this methodology for the analysis of small RNAs from EVs is described in detail elsewhere30. Analysis of our sRNAseq data determined that there are no statistically-significant differences for total reads obtained and reads passing filters  analysis for small RNAs (sRNAseq) on all EV-RNA samples as described in the Methods section. In the present study we chose to use the R package derfinder2FC >  ± 1.0). Overall, sRNAseq analysis of small RNA extracted from Vn96-isolated EVs permits the separation of PDAC patients from non-affected individuals, with each group having a unique EV small RNA expression profile.We then performed an unsupervised hierarchal clustering analysis using all 7408 regions, which generated the hierarchical clustering and heat map shown in Fig. We next performed unsupervised hierarchal clustering analyses using each individual annotation category for the analysis, including miRNA, piRNA, tRNA, mRNA (fragments), and lncRNA (fragments). These analyses provide an understanding of the utility of each small RNA species for segregating PDAC patients from non-affected individuals.32 and have been specifically investigated for the detection and/or diagnosis of pancreatic cancer, both in PDAC tissue samples34 and EVs isolated from PDAC patients12. We were therefore interested in determining whether miRNAs contained within EVs isolated with the Vn96 synthetic peptide could also permit PDAC detection. Unsupervised hierarchal clustering analysis of the 510 miRNAs identified by our sRNAseq permitted segregation of the majority of PDAC patients from non-affected individuals . Using the 659 piRNAs for clustering analysis we were able to poorly segregate the PDAC patients from non-affected individuals  highest fold change in PDAC patients versus non-affected individuals and/or 2) present in PDAC patients but absent in non-affected individuals, and/or 3) supported by the literature as a diagnostic or novel biomarker for PDAC. We therefore selected for further study miR-21, for which high expression levels in PDAC tumours have been shown to predict poor overall survival44; miR-150, whose decreased expression in PDAC tumours is associated with higher mortality45; miR-374b, which has been shown to contribute to cisplatin resistance in PDAC when its expression is decreased46; and miR-744, whose high expression in plasma has been correlated with lymph node metastasis and poor prognosis in PDAC patients9.We continued our investigation into differentially-expressed miRNAs found in Vn96-isolated EVs from the plasma of PDAC patients by selecting a few for follow-up validation and study. We chose to perform these experiments using droplet digital PCR (ddPCR), which allows for more accurate and reproducible measurements of the absolute copy number of miRNAs (via measurement of cDNA content) and unlike quantitative RT-PCR does not require normalization, and is thereby better suited for clinical assaysWe generated cDNA from the EV-RNA of our 13 non-affected individuals and 16 PDAC patients, which was then subjected to ddPCR analysis. Compared to non-affected individuals, all miRNAs that were found to be increased or decreased in PDAC patients’ EVs by sRNAseq were validated by ddPCR. The average miRNA copy number is significantly higher in PDAC patients for miR-21  allows for the discrimination of PDAC patients from non-affected individuals. Overall, our findings align with previously-published studies on small RNA species that are associated with PDAC and provide a basis for future investigations into the utility of EV-specific small RNA species for the detection of PDAC.We set out to determine whether the differential expression of small RNA species in plasma EVs isolated with the Vn96 synthetic peptide could distinguish between pancreatic ductal adenocarcinoma (PDAC) patients and non-affected individuals. This study used derfinder for the analysis of small RNA sequencing (sRNAseq) data from EV-RNA, and our findings demonstrate the utility of this approach for analyzing EV-RNA sequencing datasets. As opposed to more common analysis approaches for sRNAseq data generated from EVs, such as exceRpt49). Many of these studies have focused on EV-RNA as a biomarker source and have found that differential expression analysis of specific miRNAs can differentiate between PDAC and non-affected individuals. In agreement with this, we have also found that differential expression of miRNA in EVs captured with the Vn96 synthetic peptide distinguishes between PDAC and non-affected individuals . Approval for this study was obtained from the Research Ethics Board of the Vitalité Health Network and written informed consent was obtained from the patients. Whole blood was processed, where possible, within 30 min of collection by centrifugation at 1500×g for 15 min at 4 °C to separate the plasma fraction and stored at − 80 °C until use. Characteristics for all blood donors are shown in Table Human plasma derived from whole blood collected in EDTA-tubes was acquired from the Tumoral Biobank at the Georges-L. Dumont University Hospital Centre  and the Ontario Pancreas Cancer Study at Mount Sinai Hospital . All methods were carried out in accordance with the Canadian Research Tri-Council policy on ethical conduct for research involving humans , following established protocols24. Briefly, pre-cleared plasma (2–4 mL) was diluted 1:1 with 1 × RNase-free phosphate buffered saline (PBS) and 150 µg of Vn96 peptide added per mL of starting plasma, then incubated with end-over-end rotation for 1 h at room temperature. Vn96-EV complexes were sedimented by centrifugation at 17,000×g for 15 min, then washed three times with 1 mL PBS. RNA was extracted with the miRVana miRNA Isolation kit following manufacturer’s protocol for total RNA isolation. The yield (ng per mL of starting plasma) and total RNA profile was assessed on a Fragment Analyzer System (Agilent Technologies Inc.).Plasma was thawed at room temperature and pre-cleared at 3000×26. Briefly, small RNA libraries were prepared with 8–25 ng of RNA using the CleanTag Small RNA Library Prep kit (TriLink BioTechnologies). Raw reads from sRNAseq were processed with the Torrent Suite Software (v5.4.0), which runs the Torrent Mapping Alignment Program (TMAP). The output was converted to fastq files and the adapter sequences (TCACCGACTGCCATAGAG) were removed using TrimGalore . Alignment was performed using STAR60 (version 2.7.0f.) and the GRCh38 human genome with the parameters “–outFilterScoreMinOverLread 0 –outFilterMatchNmin 16 –outFilterMatchNminOverLread 0 –outFilterMismatchNoverLmax 0.05 –alignIntronMax 1 –alignEndsType EndToEnd”. The resulting .bam files were passed through a counting method by chromosome location using the Bioconductor package derfinder27 (version 1.18.9). Finally, the chromosome positions with counts were annotated using multiple databases such as Gencode61 (v.38), pirnaDB62 (v.1.7.6), MINT tRNA fragment database63 (v.2.0), and Mirbase64 (v.21). R statistical environment (v3.4.1) was used for the statistical analysis of the data. Data was filtered, keeping only the chromosome locations with more than 10 reads in at least 7 non-affected individuals’ samples or 8 PDAC patients’ samples. The variance between the normalized expressed chromosome regions read counts was calculated with trimmed mean of M-values (TMM) normalization method and differential expression analysis was performed using the Bioconductor package edgeR65 (v3.18.1). R was used to build the following complementary figures: heatmaps were built using heatmap.2 function from ‘gplots’ with standard parameters; including the clustering (complete) and distance methods (Euclidean) with default parameters; upset plots with upset function from ‘upsetR’; MDS plots were built with plotMDS function from ‘edgeR’ library and ggplot from ‘ggplot2’ library; and the volcano plots were built using ggplot from ‘ggplot2’library. The sRNAseq data have been deposited in the NCBI Gene Expression Omnibus (GEO) under the accession number GSE221185.Small RNA sequencing (sRNAseq) was performed as previously described26. Briefly, a volume of 5.5 µL of diluted cDNA was added to 11 µL of QX200 EvaGreen 2 × Supermix, 2.2 µL of forward primer, 1.1 µL of miScript Universal primer and 2.2 µL of nuclease-free water. Droplets were generated by mixing 20 µL reactions with 70 µL of QX200 droplet generation oil for Eva Green on a QX200 droplet generator instrument (Bio-Rad). Cycling protocol consisted of an initial 95 °C for 5 min, then 44 cycles of 95 °C for 30 s, 56 °C for 1 min and 72 °C for 2 min, then 5 min at 4 °C and 5 min at 90 °C. Ramp speed was set to 2 °C/s. Droplet fluorescence was detected on a Qx200 Droplet Reader instrument (Bio-Rad). A minimum of 10,000 droplets needed to be detected for the sample to be valid for analysis. miScript Primer Assays (Qiagen) were used for all miRNA of interest. To account for any pipetting error, samples were run in duplicate and the average copies/µL of both wells calculated. Data are expressed as the number of miRNA copies per RNA input.RNA (1 ng) was reverse transcribed into cDNA using the miScript II RT kit (Qiagen) following manufacturer’s protocol. Resulting cDNA was diluted 1:8 with DNase-free water and used as a template for droplet digital PCR (ddPCR). The QX200 ddPCR system from Bio-Rad Technologies was used to measure the concentration (copies/µL) of specific miRNA as previously describedStatistical differences between conditions were evaluated using two-Sample t-test with GraphPad Prism (v9).Supplementary Information.