The tetrel bond strength for the [I4C···X−] series and [I4Tt···X−] , was weak-to-moderate, whereas that in the remaining 16 complexes was dative tetrel bond type with very large interaction energies and short Tt···X close contact distances. The basis set superposition error corrected interaction energies calculated with the highest-level theory applied, [CCSD(T)/def2-TZVPPD], ranged from −3.0 to −112.2 kcal mol−1. The significant variation in interaction energies was realized as a result of different levels of tetrel bonding environment between the interacting partners at the equilibrium geometries of the complex systems. Although the ωB97X-D computed intermolecular geometries and interaction energies of a majority of the [I4Tt···X−] complexes were close to those predicted by the highest level of theory, the MP2 results were shown to be misleading for some of these systems. To provide insight into the nature of the intermolecular chemical bonding environment in the 25 molecule–anion complexes investigated, we discussed the charge-density-based topological and isosurface features that emanated from the application of the quantum theory of atoms in molecules and independent gradient model approaches, respectively.Twenty-five molecule–anion complex systems [I Ion–molecule interactions are fascinating in chemistry ,2,3,4, b−, Br−, or I−) attracts the electrophiles on bonded Si atoms in the neutral molecules. Neither anion is located precisely at the centroid of the neutral molecule(s). When the anion (Cl−) is entrapped inside the Si20 cage of a fully or partially chlorinated icosasilane molecular entity (20 cage to stabilize the tetrel-centered (Cl−)Si···Cl and/or (H−)Si···Cl close contacts.r entity e,f, its The fundamental phenomena that drive isolated neutral molecules to self-assemble with anions play a significant role in the processes of anion recognition and anion transport, among others ,17,18. O4, of the tetrel tetrahalide family (TtX4), where Tt stands for the elements in Group 14 of the periodic table and X represents the halide derivative . The anions considered were the halide derivative, X−. It is worth mentioning that the theoretical chemistry of 1:1 complexes formed of lighter members of the TtX4  family with the first three halide anions was recently reported  complexes be called ordinary tetrel bonds . Density functional theory  ,31,32,33 (QTAIM) ,41,42,43 (QTAIM) ,45-based (QTAIM) ,46 respo(ωB97X-D ) and ab  methods, in conjunction with the def2-TZVPPD basis set, were also employed to demonstrate the accuracy of MP2 and ωB97X-D geometries and energies of [I4Tt···X−]. Standard non-relativistic calculations were performed without considering the effect of spin-orbit coupling for heavy atoms such as Pb, following a previous study  level. An isoelectron density envelope of 0.001 a.u. was used on which to compute the potential, even though the use of higher isoelectron density envelopes was suggested in other studies for chemical systems containing low-polarizable atomic basins  are consistent with the topological charge-density-based features emanated using QTAIM. Both Multiwfn . The AIMAll code was used  and [ωB97X-D/def2-QZVPPD]. The best agreement between experiment and theory is observed with [ωB97X-D/def2-QZVPPD], and MP2 shows a tendency to underestimate r(Tt–I). A very similar trend was obtained with these methods in conjunction with the def2-QZVPPD basis set.Crystals of TtIe (ICSD) ,78,79,804. From these, it may be seen that the atomic basins are connected to each other in each isolated monomer through bond paths (solid lines between atomic basins in atom color) that pass-through bond critical points (tiny green spheres), thus recovering the expected tetrahedral Td shape of TtI4.b is larger at the C–I bcps in CI4 than at the Pb–I bcps in PbI4. It follows the trend across the series: ρb (C–I) > ρb (Si–I) > ρb (Ge–I) > ρb (Sn–I) > ρb (Pb–I). The trend signifies that the charge concentration is predominant at the C–I bcps in CI4 relative to that at the Pb–I bcps in PbI4.The charge density ρb, b is stabilizing (Hb < 0) at the Tt–I bcps, which is due to the potential energy density Vb that dominates over the gradient kinetic energy density Gb at the bcp. Hb is increasingly more positive at the Tt–I bcps across the series from CI4 through SiI4 to GeI4 to SnI4 to PbI4. This is consistent with the character of Tt–I bonds in TtI4, in which it progressively becomes less covalent than ionic, passing from CI4 through SiI4 to PbI4. That is, the covalency of the Tt–I bond follows this order: C–I > Si–I > Ge–I > Sn–I > Pb–I. Furthermore, the sign of ∇2ρb at Tt–I bcps is also negative for all monomers except for the Tt–I  bcps, giving an indication that the Tt–I bonds in CI4, SiI4, and GeI4 are relatively more covalent than those in SnI4 and PbI4. Typically, ∇2ρb < 0 and Hb < 0 represent covalent (shared-type) interactions; ∇2ρb > 0 and Hb > 0 represent ionic (closed-shell) interactions; and ∇2ρb > 0 and Hb < 0 represents mixed  interactions  complexes. They are shown in The five halide anions have linearly approached the Tt atom from the opposite side of the I–Tt covalent bond in TtI4 with the five halide anions. Because the σ-hole on the carbon atom in CI4 is the weakest compared to that on the Tt atom in TtI4   , the strC···At−] , respect4C···X−] increases as the halogen derivative becomes more polarizable; it is smallest in [I4C···F−], with r(Tt···F) = 2.665 Å with [CCSD/def2-TZVPPD]. By contrast, the I–C–I angle in complexed [I4C···F−] either increases or decreases compared to that of its uncomplexed counterpart (∠I–C–I = 109.47°). For instance, the I–C bond linearly attached to the anion forms smaller ∠I–C–I with the three nearest-neighbor I atoms, whereas the remaining I–C bonds that are not directly involved in the formation of tetrel bond are associated with larger ∠I–C–I. These two angles types are 107.5 (111.3°), 108.1 (110.8°), 108.4 (110.5), 108.8 (110.1°), and 108.8 (110.1°) in [I4C···F−], [I4C···Cl−], [I4C···Br−], [I4C···I−], and [I4C···At−], respectively.The C···X intermolecular distance in [I4C···F−], 4C···F−]. The latter ones mimic the I···X  links in the other four members of the same family , a feature that has been recommended for identifying hydrogen bonds [4C···F−], [I4C···Cl−], [I4C···Br−], [I4C···I−], and [I4C···At−] are ca. 2.871, 3.626, 3.892, 4.274, and 4.322 Å, respectively. The former three are less than their respective sum of vdW radii of 3.50 (I + F), 3.86 (I + Cl), and 3.90 Å (I + Br), whereas the latter two are slightly greater than the sum of their respective sum of vdW radii of 4.08 (I + I) and 4.04 Å (I + At). . Since rom ref. ,62,97,99re known ,101, whi4C···X−], as CCSD. However, the increase in the size of the basis set from def2-TZVPPD to def2-QZVPPD has resulted in a slight increase in the Tt···X intermolecular distance with ωB97X-D and MP2. Whatever is the size of the basis set, the intermolecular distances predicted using MP2 are underestimated relative to ωB97X-D and CCSD. Furthermore, [MP2/def2-TZVPPD] has predicted the C···I and C···At bond distances to be 4.091 and 4.083 Å for [I4C···I−] and [I4C···At−], respectively; they were 3.990 and 4.009 Å with def2-QZVPPD, respectively. This means that MP2 does not correctly predict the trend in C···I and C···At bonding distances, as predicted by ωB97X-D and CCSD  and Hb (Hb < 0) at the bcps of the three equivalent bonds, were equivalent  > [I4C···Cl−] (δ = 0.030) > [I4C···Br−] (δ = 0.023) > [I4C···I−] (δ = 0.019) > [I4C···At−] (δ = 0.017), in consistent with the trend found for interaction energy  is completely lost.The nature of the intermolecular bonding environment found in  c–e. Simil, or Br a–c, but Ge···X−] d,e. This4Si···Br−], in which, the responsible interacting units were involved in the formation of a dative tetrel bond; it is in a manner similar to that found for [I4Si···X−] . On the other hand, MP2 has recognized the attraction between I4Si and X− in the first four members of the [I4Si···X−] series to be unusually strong and that in [I4Si···At−] to be moderate. The former result with MP2 is applicable to the [I4Ge···X−] series as well. This means that the Tt···X close contacts in the above-mentioned molecule–anion systems are not ordinary tetrel bonds; they are dative tetrel bonds.While the bonding features noted above were obtained from [ωB97X-D/def2-QZVPPD], the [CCSD/def2-QZVPPD] method has predicted an exception for [Ib is appreciable at the Ge···X bcps in [I4Ge···X−] when X points to F, Cl, and Br. For [I4Ge···I−], the ρb is small at Ge···I bcp (ρb = 0.0048 a.u.), and the interaction between the monomers is also reinforced by I···I interactions  bcps in [I4Ge···X−] are not only typical for coordinate bonds but larger than that can be expected for ordinary non-covalent interactions such as hydrogen bonds, and halogen bonds, among others (ρb < 0.05 a.u.). They may be comparable with the ρb values of the Tt–I coordinate bonds in isolated and complexed TtI4. A similar conclusion might be arrived at for Si···X bcps in [I4Si···X−] , Our QTAIM analysis, ractions d. The ρb2ρb (∇2ρb > 0) and Hb (Hb < 0), 4Si···X−] provided [CCSD/def2-TZVPPD] results are considered. The large δ values corresponding to atom–atom pairs responsible for the Si···X and Ge···X  close contacts provide further evidence that there are no π-type interactions involved; they are purely σ-type coordinate dative bonds. By contrast, the δ values are very small for atom–atom pairs causing the Si···X and Si···X close contacts in [I4Ge···X−] and [I4Ge···X−] , respectively, indicative of closed-shell interactions. The three equivalent I···X halogen···halogen close contacts in each of [I4Ge···X−] and [I4Ge···X−]  are described by small δ values, and positive ∇2ρb and Hb. Similarly, the Si···X and Ge···X  tetrel bonds are described by small δ values, as expected.From the sign and magnitude of ∇4 and PbI4, respectively, are stronger than those of TtI4 . Therefore, their acidic strengths are adequate enough to recognize the five halide anions when in close proximity. This may be rationalized from QTAIM’s molecular graphs of resulting configurations, [I4Sn···X−] and [I4Pb···X−] , illustrated in 4 and PbI4. This means that the TtI4 molecule is structurally fully deformed in the presence of each of the five halide anions. There is no secondary intermolecular interaction that can play a role in the geometrical stability of the resulting complex anions, as found for other series (see above). In all cases, the tetrel center adopts a trigonal bipyramidal geometry . Clearly, the resulting complex anions each is nothing but a coordination compound, and the Tt···X close-contact is formally a Tt–X dative tetrel bond. In such cases, charge transfer from the anion to the σ*(I–Tt) anti-bonding orbital is expected, and the SN2 mechanism is likely to play a role in driving the dative bond formation between the interacting species [The σ-holes on Sn and Pb in SnI species .4Pb···X−] series and the smallest for the [I4C···X−] series. In particular, the [ωB97X-D/def2-QZVPPD] level QTAIM charge transfer from X− to PbI4 is 0.263, 0.371, 0.424, 0.515 and 0.588 e for [I4Pb···F−], [I4Pb···Cl−], [I4Pb···Br−], [I4Pb···I−], and [I4Pb···At−], respectively. The corresponding charge transfer values were 0.229, 0.334, 0.382, 0.460, and 0.519 e for [I4Sn···F−], [I4Sn···Cl−], [I4Sn···Br−], [I4Sn···I−], and [I4Sn···At−], respectively. These results imply that the nature of charge-transfer in the Sn- and Pb-based anions is virtually similar and that the charge-transfer preference is consistent with the interaction energy preference across a given series, indicating that the charge-transfer phenomenon is likely to be one of the most prominent contributors to the interaction. Our calculation suggests that the extent of charge transfer is the largest for the [Ie calculated for [I4C···F−], [I4C···Cl−], [I4C···Br−], [I4C···I−], and [I4C···At−], respectively. These results may lead to a conclusion that the formation of stronger complexes accompanies an appreciable amount transfer of charge between the interacting monomers, and is not very surprising   is non-negligible; it may be comparable to that of the Tt–I bcps of complexed I4Tt. The Tt–I and Tt···X bcps are both characterized by ∇2ρb > 0 and Hb < 0, indicating the presence of a mixed covalent and ionic character. Since Hb becomes increasingly more positive at the Tt···X bcp passing from [I4Tt···F−] through [I4Tt···Cl−] to [I4Tt···Cl−] to [I4Tt···Br−] to [I4Tt···I−] to [I4Tt···At−], it is clear that these interactions are less covalent in the same order. The ∇2ρb values are decreasing in the series from [I4Tt···F−] through [I4Tt···Cl−] to [I4Tt···At−], indicating that the Tt···F tetrel bond is more ionic than the Tt···At tetrel bond. In the case of Tt = Si and Ge, the Tt···X  bcps show ∇2ρb > 0 and Hb < 0  bond in the respective system. The characteristics of Si–I bonds in I4Si resemble the C–I bonds in I4C.From the molecular graphs of QTAIM in d Hb < 0 . The fou4Tt···X−]  is considerably larger than what were calculated for ordinary tetrel bonds  upon its attractive engagement with the halide anions. For comparison, we note that the δ values for Tt–I and Tt···X coordinate and dative tetrel bonds in [I4Tt···X−] are smaller than, and comparable to, those reported for metal–C(O) coordinate bonds ; however, those of Tt···X ordinary tetrel bonds are comparable with what were reported for metal···metal  interactions  in [M2(CO)10] and [M3(μ-H)3(CO)12]  complexes  (Tt = C). The I···X closed contacts in several of these systems appeared at larger IGM-interδg isovalues (Top). On the other hand, the Tt···X close contacts showed up at lower IGM-interδg isovalues (Bottom). This is not very surprising since smaller isolvalues are typically necessary for the physical appearances of isosurfaces corresponding to weakly bonded interactions. By contrast, the relatively stronger interactions can be traceable with larger isovalues since charge density around the critical bonding region is generally appreciable. The bluish isosurface originated with large IGM-interδg isovalues for [I4C···F−] indicates that the attraction between the interacting units is very prominent . The view is also transferable to the [I4Ge···X−] systems (not shown).The [I4Sn···X−] and [I4Pb···X−] , the IGM-interδg isosurfaces were visualizable with an isovalue close to 0.055 a.u. 4Pb···X−] series. Passing from the left to the right of 4Tt···F−] through [I4Tt···Cl−] to [I4Tt···Br−] to [I4Tt···I−] to [I4Tt···At−] .In the case of [I4C···X−] series and [I4Tt···X−] , the int (BSSE)E values for all other molecule–anion systems are much larger than the so-called covalent limit for hydrogen bonds  [4Tt···X−], it is clear that the complex stability is largely determined by the polarizability of the Tt atom in I4Tt and the halogen derivative. These energies calculated in the range from −3.0 to −112.2 kcal mol−1 with [CCSD(T)/def2-TZVPPD], −1 < int (BSSE)E < −5.0 kcal mol−1), moderate E < −10.0 kcal mol−1), strong E < −25.0 kcal mol−1), very strong E ≤ −40.0 kcal mol−1), and ultra-strong (int (BSSE)E >> −40.0 kcal mol−1 . At the highest level of theory applied, [CCSD(T)/def2-TZVPPD], the weakest and strongest of the [I4Tt···X−] systems are found to be [I4C···At−] (int (BSSE)E = −3.20 kcal mol−1) and [I4Si···F−] (int (BSSE)E = −112.15 kcal mol−1), respectively.Except for the  with def2-TZVPPD and def2-QZVPPD, respectively; these are indicative of the fact that the strength of the tetrel bond between Si of I4Si and Br− is moderate. As mentioned already above, this is not the case with MP2 since the intE (BSSE) for the same system, for instance, with def2-QZVPPD, is predicted to be −60.0 kcal mol−1; the large intE (BSSE) implies that the attraction between I4Si and Br− causes the formation of Si–Br dative tetrel bond. This result is consistent with [CCSD(T)/def2-TZVPPD], which has predicted an intE (BSSE) of −53.16 kcal mol−1 for the same system. Second, the [ωB97X-D/def2-TZVPPD] level intE (BSSE) values for the remaining four systems of the [I4Si···X]− series are in qualitative and quantitative agreement with [CCSD(T)/def2-TZVPPD]. MP2, however, unusually overestimated the interaction energies for [I4Si···I−] and [I4Si···At−]. The discrepancy between the DFT (or CCSD(T)) and MP2 energies is likely due to the latter method’s misleading prediction of the Si···I and Si···At close contacts, thus pushing the interacting atoms in [I4Si···I−] to be bonded with each other via a dative tetrel bond. These peculiar results indicate that applying the MP2 approach to predict the correct nature of the tetrel bond in molecule–anion complex systems formed by heavier tetrel derivatives in molecular entities should be exercised with caution.From int(BSSE),E between the five members of each series [I4Tt···X]− follows the trend: [I4Tt···F−] > [I4Tt···Cl−] > [I4Tt···Br−] > [I4Tt···I−] > [I4Tt···At−] . Note that changing the basis set from def2-TZVPPD to def2-QZVPPD somehow restored the [CCSD(T)/def2-TZVPPD] level energy preference at the ωB97X-D level (but not at the MP2 level) when Tt = C, but not when Tt = Pb. One reason for the anomalous change in the preference of energy ordering between [I4Tt···I−] and [I4Tt···At−] is that the post-HF MP2 method greatly overestimates the BSSE, as well as the electron-electron correlation energy, relative to the DFT and CCSD(T). On the other hand, the CCSD(T) method has properly accounted for electron-electron correlation energy, which ensured the correct preference of stabilization energies among the five members of any given series, [I4Tt···X−].The preference of BSSE-corrected interaction energy, t···At−] . This isint(BSSE)E on the distance of separation r(Tt···X) for 25 molecule–anion complexes, [I4Tt···X−], obtained using ωB97X-D, MP2 and CCSD(T). Regardless of the different calculation methods utilized, the dependence was found to be quadratic. The square of the regression coefficient R2 was moderately higher (R2 = 0.9325) for ωB97X-D compared to CCSD(T) and lower (R2 = 0.8923) for MP2.−1, from 2.60 to 13.58 kcal mol−1, and from 1.42 to 13.44 kcal mol−1, respectively. However, when using the def2-QZVPPD basis set, the BSSE in energy has decreased sharply, giving rise to values in the range from 0.02 to 0.13 kcal mol−1 with ωB97X-D and from 1.01 to 5.01 kcal mol−1 with MP2. CCSD(T) with def2-QZVPPD was computationally very expensive; no conclusions could be drawn about the range of BSSE in energy with this method. int(BSSE)E and intE, obtained using [ωB97X-D/def2-QZVPPD], [MP2/def2-QZVPPD] and [CCSD(T)/def2-TZVPPD], respectively, showing a perfect linear dependence at the former level than that at the latter two. We note further that the BSSE in energy is minimal with DFT but larger with MP2 and CCSD(T). It is very large with the def2-TZVPPD basis set than with the def2-QZVPPD basis set. For example, for ωB97X-D, MP2, and CCSD(T) with def2-TZVPPD, the BSSE in energy ranged from 0.05 to 9.68 kcal mol4Tt···X−]  was theoretically investigated to clarify the nature of the selectivity of the I4Tt host for five guest  anions. The MP2 geometries and interaction energies for the 25 molecule–anion systems were underestimated and overestimated, respectively, relative to DFT and CCSD methods, and in some cases, the MP2 results were unreliable. The chemical bonding features obtained using DFT were consistent with the computationally expensive CCSD and CCSD(T) results, with an exception for [I4Si···Br−]. For the latter, the tetrel bonding characteristics predicted by CCSD could not be reproduced by DFT-ωB97X-D. Similarly, the significant overestimation of the interaction energy of [I4Si···I−] with MP2 was in sharp disagreement with that computed using ωB97X-D and CCSD(T).In this study, the series    systems, except for [I4C···X−]  and [I4Si···X−] . This was the case with [ωB97X-D/def2-TZVPPD] and [ωB97X-D/def2-QZVPPD], but [I4Ge···At−] was added to the exclusion list. The result was different from [MP2/def2-QZVPPD] in that it predicted exceptions only for [I4C···X−] , and is not surprising given it is an MP2’s tendency to underestimate intermolecular distances. Although these latter two computational methods exclude intermolecular interactions in systems that do not follow the stringent “less than the sum of vdW radii rule,” the exclusion was also consistent with the nature of bond path topology revealed using QTAIM. For example, for [I4C···X−] , [I4Tt···X−]  and [I4Tt···X−] , no QTAIM-based bond path topology exists between Tt and X at the [ωB97X-D/def2-QZVPPD] level. This means that QTAIM does not recognize the existence of Tt···X tetrel bonding in the host–guest systems when the tetrel bond distance between Tt and X exceeds the sum of the vdW radii of Tt and X, even though this type of limitation of QTAIM has been attributed to the arbitrary nature of the space partitioning scheme. Even so, it should be borne in mind that the Tt and X atoms of the interacting monomers in all complex systems were indeed tetrel bonded to each other, evidenced by the IGM-interδg-based isosurfaces.The Tt···X separation distance calculated by [CCSD/def2-TZVPPD] was smaller than the vdW radii of the Tt and X atoms for all [I


Sleepiness has been associated with cognitive decline and dementia in the elderly. Older adults with excessive daytime sleepiness appear to be more vulnerable to longitudinal amyloid PET accumulation before the onset of the dementia. However, it remains unclear whether sleepiness is similarly associated with other biomarkers of Alzheimer’s disease (AD), axonal integrity, and inflammation, which may also contribute to neurodegeneration and cognitive decline.In this cross-sectional analysis, we identified 260 cognitively unimpaired adults (>60 years) from the Mayo Clinic Study of Aging, a population-based cohort from Olmsted County (MN), who underwent CSF quantification of AD biomarkers  in addition to at least one of the following biomarkers . We fit linear regression models to assess associations between sleepiness, as measured by the Epworth Sleepiness Scale (ESS), and CSF biomarkers, controlling for age, sex, APOε4 status, body mass index, hypertension, dyslipidemia, and prior diagnosis of obstructive sleep apnea.p = 0.033) increase in the log of IL-6 and 0.01  increase in the log of NfL. A sensitivity analysis showed an association between ESS scores and log of p-tau/Aβ42 only in participants with an abnormal ratio (>0.023), highly predictive of amyloid positivity. For every ESS score point increase, there was a 0.006  increase in the log of CSF p-tau/Aβ42.Higher ESS scores were associated with higher CSF IL-6 and NfL, but not with the other CSF biomarkers. For every ESS score point increase, there was a 0.009 ([95% CI 0.001–0.016], via disturbed connectivity or damage to wake-promoting centers. Associations between sleepiness and p-tau/Aβ42 in participants with abnormal ratio suggest that amyloid positivity contributes to vulnerability to sleep disturbance, which may further amyloid accumulation in a feed-forward loop process. Prospective studies of these markers are needed to determine cause-effect relationships between these associations.Sleepiness was associated with greater CSF IL-6 and NfL levels, which could contribute to neurodegeneration or alternatively cause sleepiness. Higher NfL levels may result from sleep disruption and/or contribute to sleepiness  A meta-analysis of 27 observational studies estimated that approximately 15% of Alzheimer’s disease (AD) in the population may be associated to sleep disturbance . Sleepinin vivo pathology in pre-clinical stages of AD (We have previously shown that excessive daytime sleepiness (EDS) in older adults was associated with global cortical thinning, suggestive of accelerated brain aging . We alsoes of AD .Pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), have been implicated in AD  and neurThe objective of this study was to identify associations between sleepiness in CU older adults with (1) CSF biomarkers of AD pathology ; (2) CSF biomarkers of neuroinflammation ; and (3) a CSF biomarker of axonal integrity and neurodegeneration (NfL).n = 251–260 depending on the biomarker) and completed the Epworth Sleepiness Scale (ESS) as part of the clinical assessment. There were no other exclusion criteria.The Mayo Clinic Study of Aging (MCSA), which began in 2004, is a population-based cohort of residents living in Olmsted County . This study was approved by the Mayo Clinic and Olmsted Medical Center institutional review boards and informed consent was obtained from all participants or their surrogates. Details of the MCSA design have been published elsewhere . For theCognitive status was determined by a consensus committee including the study coordinator, neuropsychologist, and the physician who evaluated each participant, as previously described . ParticiParticipants completed the ESS  for asseAPOε genotyping was performed as previously described . ParticiHistory of medical conditions was abstracted by trained nurses using the Rochester Epidemiology Project (REP) medical records-linkage system . Body maCSF was obtained through lumbar punctures performed early in the morning in the lateral decubitus position. The sample was divided into 0.5-mL aliquots and stored at −80°C for future analyses avoiding freeze–thaw cycles prior to the current analyses. CSF Aβ42 and tau phosphorylated at threonine 181 (P-tau181) was measured with automated electrochemiluminescence Elecsys immunoassays (Roche Diagnostics) at Mayo Clinic Rochester. CSF IL-6, IL-10, and TNF-α levels were measured on the Simoa HD-1 platform . The CSF NfL levels were measured in the Clinical Neurochemistry Laboratory at the University of Gothenburg using an in-house sandwich enzyme-linked immunosorbent assay (ELISA) . The levp-value < 0.05 was considered statistically significant.We performed cross-sectional analyses to examine associations between CSF biomarkers and sleepiness, as measured by ESS scores. Due to non-Gaussian or skewed distribution of CSF biomarkers, these variables were analyzed with logarithmic transformation, except for TNF-α, which could not be satisfactorily normalized and required non-parametric testing. Multivariable linear regression models were fit separately with each CSF biomarker (except TNF-α) as the dependent variable. ESS score was added as our independent variable of primary interest adjusting for age, sex, APOε4 genotype carrier, BMI, hypertension, dyslipidemia, and OSA diagnosis. As an initial step, we included all variables and then used backward elimination  to obtain a final parsimonious model, less susceptible to over-fitting of data or unstable associations. Quantile regression was used to examine a possible association between TNF-α levels and ESS scores. For subgroup analyses of amyloid-positive participants or those with available AHI, unadjusted Pearson and Spearman’s rank correlations were initially performed to examine associations between two variables, according to data distribution. Subsequent analyses with either Pearson partial correlation or multiple variable linear regression were performed if initial unadjusted correlations were significant. Logarithmic transformation of variables were pursued, if necessary, to allow linear approximation. Owing to the exploratory nature of this work, adjustment for multiple comparisons were not performed. Statistical analyses were performed with SPSS software for Windows . A two-sided The sample characteristics are summarized in p = 0.033) increase in the log of IL-6 and 0.01  increase in the log of NfL. There were no associations between ESS and any of the other biomarkers . Given that amyloid status measured by amyloid PET strengthened the association between EDS and longitudinal amyloid PET changes in our previous study (p = 0.048) only in those with abnormal ratio  (p = 0.021) increase in the log of CSF p-tau/Aβ42. No associations were observed with other AD biomarkers.In multivariable linear regression analyses assessing for associations between CSF biomarkers  and ESS scores, adjusted for age, sex, APOε4 genotype status, BMI, dyslipidemia, hypertension, and prior OSA diagnosis; we found significant associations between CSF IL-6 and NfL levels with ESS scores. These associations persisted after backward selection procedure . For eveus study , we splius study . We firs n = 60) . In the b = 0 [95% CI −0.001 to 0.002], p = 0.479).A diagnosis of OSA was not associated with any of the CSF biomarkers. In addition, in a sensitivity analysis replacing either snorting/choking or witnessed apneas for OSA diagnosis, the association between ESS scores with CSF IL-6 and NfL levels remained significant. We did not find a significant association between ESS scores and median CSF TNF-α using quantile regression in the multivariable model, controlling for the same variables included in the linear models , but not with IL-6. There were significant correlations between AHI and ESS , IL-6 , NfL , and Aβ42 . However, these associations did not remain significant after we performed partial correlations adjusting for age and sex with AHI distribution normalized using common logarithmic transformation.In a subgroup analysis of 54 participants with available AHI from the original OSA diagnosis, ESS scores correlated with log of NfL after controlling for AHI  showed that sleep disturbance was associated with increased serum/plasma IL-6 levels . It appears that the relationship between IL-6, AD and neurodegeneration depend on other pro-inflammatory and anti-inflammatory cytokines and different endophenotypic expression of inflammatory markers   to promo markers , in addi markers .Neurofilament light chain may be associated with sleep disturbance, but the literature has shown contradictory findings. Middle-aged insomnia patients have higher NfL levels compared to those without insomnia , and onevia increased oxidative stress and neuroinflammation causing neurodegeneration  in 79 elderly participants without dementia (median age 76.5 [IQR 71.7–80.7])  . However7–80.7]) . Levels 7–80.7]) . We cannAlthough sleepiness was not associated with either CSF Aβ42 or p-tau levels, it was associated with p-tau/Aβ42 in patients with an abnormal ratio. This abnormal ratio has been found to be a better predictor of amyloid positivity than individual AD biomarkers . This suLimitations of the study warrant consideration. First, the cross-sectional design of this study does not allow us to determine whether the associations found are causal. For instance, we cannot determine if sleepiness is a marker of more severe sleep disturbance that leads to an increase in neuroinflammation, axonal damage, AD pathology, and neurodegeneration; or if an abnormal neuroinflammatory response, connectivity issue caused by axonal injury, and/or neurodegeneration of wake-promoting centers results in more sleep disturbance and/or daytime sleepiness. Larger prospective longitudinal cohort studies will be necessary to determine the directionality of these associations. Second, we relied on self-reported sleepiness as opposed to objective measures of sleepiness , which might have obscured some associations. Older individuals may underestimate their sleepiness as measured by ESS. In a cross-sectional observational study including 104 independently living non-demented older subjects with daytime sleepiness complaints and 104 non-demented close relatives (CRs), subjects rated their sleepiness with ESS scores significantly lower (7.10 ± 4.31) than their CR proxy did (9.70 ± 5.14) . HoweverOur study suggests a complex relationship between sleepiness and CSF IL-6 and NfL. Higher IL-6 and NfL may be associated with sleepiness either as a cause or consequence of underlying sleep disturbance. Both may be associated with hypoxemia, which should be further assessed in future studies. It is also possible that higher NfL suggests greater underlying axonal injury and neurodegeneration that contributes to daytime sleepiness by disrupting attention networks or output from wake-promoting regions. Although there is evidence that both IL-6 and NfL contribute to neurodegeneration, the relationship with AD pathology is more consistent for NfL than IL-6, likely given IL-6 complex interactions with other inflammatory markers and receptor polymorphisms. Our results corroborate previous findings indicating that elderly individuals with daytime sleepiness are more likely to have greater AD pathology, and raise the possibility for increased vulnerability to neuroinflammation, neurodegeneration, and axonal injury.https://ras-rdrs.mayo.edu/Request/IndexRequest. The request will be reviewed by the Mayo Clinic Study on Aging investigators and Mayo Clinic to verify whether the request is subject to any intellectual property or confidentiality obligations. A data sharing agreement must be obtained prior to release.Raw and analyzed de-identified data from the Mayo Clinic Study on Aging can be requested using the following link: The studies involving human participants were reviewed and approved by the Mayo Clinic and Olmsted Medical Center Institutional Review Boards. The participants provided their written informed consent to participate in this study.DC, PV, and MiM: study concept and design. All authors: data collection or interpretation and critical revision of the manuscript for important intellectual content. DC: drafting of the manuscript. DC and SP: statistical analysis. All authors agreed to be accountable for the content of the work.ES has received research support from Mayo Clinic CCaTS, NIH, the Michael J. Fox Foundation, and Sunovion, Inc. MaM receives research funding from the NIH. BB has served as an investigator for clinical trials sponsored by Alector and Biogen. He serves on the Scientific Advisory Board of the Tau Consortium. He receives research support from the NIH the Mangurian Foundation, the Little Family Foundation, and the Ted Turner and Family Foundation. RP consults for Roche, Inc., Merck, Inc., Genentech, Inc., and Biogen, Inc., GE Healthcare and receives royalties from Oxford University Press for the publication of Mild Cognitive Impairment. CJ consults for Lily and serves on an independent data monitoring board for Roche but he receives no personal compensation from any commercial entity. JG-R receives research funding from NIH and serves on the editorial board of Neurology. PV receives research funding from NIH (NIA and NINDS). MiM served as a consultant to Biogen, Brain Protection Company, and LabCorp and receives research support from the NIH and DOD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Long non-coding RNAs have been reported to be involved in tumorigenesis and progression through different regulatory mechanisms. It has been reported that aberrantly expressed long non-coding RNA LINC00491 promotes malignancy in multiple tumors, while the role of LINC00491 in lung adenocarcinoma (LUAD) is little reported and the mechanism for regulating tumor progression has not been elucidated. Methods: RNA sequencing and the TCGA database were combined to screen differentially expressed lncRNAs that facilitate tumor progression. The expression level of LINC00491 was examined in LUAD clinical samples and in cell lines using RT-qPCR. In vitro experiments including colony formation assay, EdU assay, cell migration and invasion assay and wound healing assay, and in vivo experiments including xenografting subcutaneous tumors and lung metastasis models were performed to investigate the function of LINC00491 in LUAD tumor progressions. RNA pull-down, mass spectrometry, RIP assays and truncation experiments were carried out to explore the proteins binding to LINC00491 and the specific interactions between the RNA–protein complex. Results: Our results showed that LINC0491 was significantly upregulated in LUAD and positively correlated with poor survival. High LINC00491 expression promoted proliferation, migration and invasion, and resulted in a high metastatic burden in LUAD. Using pull-down assay and mass spectrometry, MTSS1 was found binding to LINC00491, and the conducted experiments verified the direct interaction between LINC00491 and MTSS1. Meanwhile, LINC00491 was found to regulate MTSS1 degradation by promoting the MTSS1 ubiquitination level and then activating the Wnt/β-catenin-signaling pathway. LINC00491/MTSS1/β-catenin may act as a complex to facilitate tumor progression. Conclusions: In summary, our results found a novel mechanism in which LINC00491 directly interacts with MTSS1 by affecting its ubiquitination modification to promote LUAD proliferation, migration and invasion, then activating the Wnt/β-catenin-signaling pathway, demonstrating its significant role in tumor progression and suggesting that the LINC00491/MTSS1/Wnt/β-catenin-signaling pathway could serve as a potential therapeutic target for lung adenocarcinoma in the future. Lung cancer has been the second most common malignant cancer and has the highest cancer-associated mortality in the world ,2. ApproMTSS1, known as missing in metastasis (MIM), is affiliated with the inverse Bin-Amphiphysin-Rvs (I-BAR) family . PreviouFor this paper, we verified the expression of MTSS1 in large numbers of clinical samples and identified LINC00491, which regulates the ubiquitin–proteasome degradation of MTSS1, resulting in high metastasis and poor prognosis. Further experiments in LUAD cell lines and mouse models confirmed that LINC00491 facilitates cell proliferation, migration and tumor metastasis through Wnt signaling via degrading MTSS1.82 LUAD tissue samples and matched adjacent normal lung tissue samples were collected from February 2015 to March 2019 at Shanghai East Hospital . The samples were placed in liquid nitrogen immediately after resection. The matched adjacent normal lung tissue samples were collected >3 cm from the edge of tumor without tumor cells. Diagnosis and grading of all cases were confirmed by three experienced pathologists. A summary of the clinicopathological features of LUAD patients is provided in A549, H1299 and H1975 human ADC cell lines and BEAS-2B human bronchial epithelial cells were obtained from the Chinese Academy of Science . These cell lines were cultured in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 15% fetal bovine serum (Gibco) and 1% penicillin-streptomycin solution (Gibco), under 5% CO2 and at 37 °C in a chamber.LINC00491 and MTSS1 were designed and synthesized by GenePharma Co., Ltd. . ShRNA plasmids for targeting LINC00491 together with their nonspecific control shRNA, and plasmids targeting LINC00491 FL, LINC00491 ∆1, LINC00491 ∆2 and LINC00491 ∆3 were designed and purchased by GenePharma Co., Ltd. After transfection for 48 h, the transfection efficiency of cells was evaluated using RT-qPCR. The sequences of shRNAs were as follows: LINC00491-sh1, 5′-GGATATGTGCAGGGAGTCTAG-3′; LINC00491-sh2, 5′-GGTGTATTCCACATT GTCTCT-3′.Lentiviral constructs overexpressing RNA isolation, reverse transcription and quantitative reverse transcription–polymerase chain reaction (RT-qPCR) followed.® II 1st Strand cDNA Synthesis Kit . The cDNA templates were amplified by RT-qPCR using a SYBR Green Master Mix . GAPDH was applied as an internal control. The primer sequences used were as follows: LINC00491-F, 5′- AAATACCACCACCCCATC-3′; LINC00491-R, 5′- GGAAACATTCTGCCTCCT-3′; MTSS1-F, 5′-CAGTCCCAGCTTCGGACAAC-3′; MTSS1-R, 5′- TGAGAGCAGATCCAATCTCCC-3′; GAPDH-F, 5′-GAGTCAACGGATTTGGTCGT-3′; GAPDH-R, 5′-TTGATTTTGGAGGGATCTCG-3′. The 2−∆∆CT method was adopted to analyze the relative expression.RNA from cell lines and tissue samples was extracted using Trizol reagent  according to the manufacturer’s instructions. The RNA from the cytoplasm contents and nucleus was isolated and extracted utilizing a PARISTM Kit . Then, RNA samples were reverse-transcribed into cDNA using a HiScriptA total of 1500 cells were seeded per well in 6-well plates and the culture medium was refreshed every 4 days. After 2 weeks, the colony cells were fixed using 4% paraformaldehyde for 20 min at room temperature and then stained with 0.1% crystal violet for 30 min. The number and size of clones were evaluated to compare the proliferation ability of LUAD cells in groups.4 cells were seeded into the upper chambers of transwell inserts  with or without Matrigel (Corning) containing 200 µL of serum-free DMEM, and 500 µL DMEM containing 10% FBS was put in the lower chambers. After 24 h, the filters were washed with PBS three times, fixed with 4% paraformaldehyde, and then stained with crystal violet. The number of cells was counted to evaluate the migration and invasion ability of cells.A total of 5 × 10Monolayers were scratched with a 200-µL pipette tip across the center of wells when cells reached 90% confluence. Then, cells were cultured in the serum-free medium and imaged at 24 h intervals to record wound width. ImageJ software was adopted to quantify the wound width.The RIP assay was performed as previously described. In brief, cells were collected using an RIP lysis buffer upon reaching 90% confluence. In the meantime, a human anti-MTSS1 antibody was incubated with magnetic beads to conjugate the antibody to the magnetic beads. Collected using a magnetic separator, proteinase K was added to digest the protein. Finally, the Trizol reagent was used to extract supernatants containing RNA. Normal rabbit IgG in the RIP Kit was used as a negative control.A biotin-labelled LINC00491 mimic was synthesized and transfected into cells as previously reported . Cells w3 per well) were seeded into 96-well plates. After 24 h, the LUAD cells were incubated with 50 µM EdU under 5% CO2 and at 37 °C in a chamber for 2 h, then fixed with 4% paraformaldehyde for 15 min and permeabilized with Triton X-100. Finally, Apollo Dye Solution was used to stain the cells and Hoechst 33,342 was used to stain the nucleic acids. Images were acquired on the basis of a Leica SP5 confocal microscope .The EdU assay was performed using a Cell-light TM EdU DNA cell proliferation kit  according to the manufacturer’s instructions. Briefly, A549 and H1299 cells . Cells were fixed using 4% paraformaldehyde, and then 0.25% Triton X-100 was used to permeabilize cells. Then, LUAD cells were hybridized with labeled probes specific to LINC00491 in a reaction buffer at 37 °C overnight. Images were acquired on the basis of a Leica SP5 confocal microscope .The digoxigenin (DIG)-labeled LINC00491 probes were purchased from Servicebio . In short, the paraffin-embedded LUAD samples were deparaffinized in xylene and rehydrated via graded ethanol. Pepsin was used to digest sections for 20 min, and then sections were prehybridized overnight at 40 °C. After washing, the sections were incubated in a LINC00491-specific probe along with subsequently biotinylated mouse anti-DIG-HRP antibody, and further stained with biotinylated peroxidase. Finally, LINC00491 expression was visualized using 3,3′-diaminobenzidine (DAB) substrate and hematoxylin was used as a counterstain. 4 LUAD cells were seeded in confocal dishes . After 24 h, the cells were fixed, permeabilized and blocked for 2 h. Then the cells were incubated with primary antibodies at 4 °C for 8 h, and with secondary antibodies for another 1 h at room temperature. Then, DAPI was used to stain the nuclei. Finally, a Leica SP5 confocal microscope  was adopted to acquire images. 2 × 10The whole protein from cells and tissues was extracted with a RIPA buffer . The protein extracts were incubated with the corresponding antibody at 4 °C overnight after being subjected to 10% SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). After being washed with TBST (TBS + Tween) three times, the membranes were incubated with goat anti-rabbit IgG (H+L) or goat anti-mouse IgG (H+L) antibodies for 1 h. Enhanced chemiluminescence (ECL)  was adopted to visualize the protein gel. β-actin served as an internal control. Primary antibodies in the experiment are listed in HE staining was conducted according to the manufacturer’s instructions using a hematoxylin-eosin staining kit . For IHC staining, sections of tissue samples were incubated with the corresponding antibody overnight at 4 °C, then with HRP Polymer secondary antibodies at room temperature for 1 h. Finally, chromogen  was taken to the stain sections, and then images were gained in the microscope for analysis.2 was used to wash protein A/G magnetic beads at 4 °C for 15 min three times. Finally, protein A/G magnetic beads were transferred to a 2X SDS-PAGE sample loading buffer (Beyotime) and incubated at 95 °C for 9 min. Co-immunoprecipitated (co-IP) proteins were analyzed using immunoblot procedures. All the ubiquitin assays were performed in denaturing conditions. IP assays were carried out according to the manufacturer’s instructions using a Magnetic IP/Co-IP Kit . Briefly, cells were lysed in an NP-40 lysis buffer (Beyotime) and the lysates were centrifuged. Incubation of the above supernatant and the targeted antibody was assigned with rotation at 4 °C overnight. After being added to samples on a rotary shaker at 4 °C for 6–8 h, 1 mL wash buffer containing 0.08% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0) and 5 mM MgCl6 logarithmically growing A549 cells from different groups were injected subcutaneously into the flank of nude mice  within 100 µL PBS. The weight and volume of tumors were examined every 5 days. After 3 weeks, euthanasia was performed on mice, and the xenografting tumors were collected for IHC analysis.4 × 106 logarithmically growing A549 cells from different groups were injected via tail vein (6 mice per group). After 8 weeks, euthanasia was conducted in mice and the metastatic lung tissues were collected for HE.For the lung metastasis model, 1 × 10t-test or one-way ANOVA. Expression correlation was evaluated by means of Pearson’s coefficient correlation. The Kaplan–Meier method was adopted to plot the survival curve and the comparison was analyzed using log-rank tests. p value < 0.05 indicates statistical significance.Statistical graphs in this study were created using GraphPad Prism 8.0 software . All statistical data are expressed as the means ± S.D. Comparisons between groups were examined using two-tailed Student’s p-value of <0.05, 54 and 123 lncRNAs were differentially expressed in tissue sequence and in the TCGA  compared with those with lower LINC00491 expression levels. Taken together, the above results illustrate that LINC00491 was differentially up-regulated in LUAD patients and LINC00491 expressional levels could be an independent prognostic factor for predicting clinical outcomes in patients with LUAD.What is more, RT-qPCR assays and RNA FISH assays were performed to clear the location of LINC00491 in the cell, and the results showed that LINC00491 was largely focalized in the cytoplasm of A549 and H1299, amplified with RNA isolated from the cell nucleus and cytoplasmic contents F,G. SubsTo identify the role of LINC00491 in LUAD, short hairpin RNA (shRNA) targeting LINC00491 was transfected in A549 and H1299 to silence LINC00491 and the transfection efficiency was confirmed using RT-qPCR . Colony To explore the potential mechanism by which LINC00491 exerts its biological functions in LUAD cells, RNA pull-down assays followed by mass spectrometry (MS) were performed. Silver staining showed a protein was markedly focused around 105 kDa compared with the LINC00491 antisense A. The foPrevious studies have proved that MTSS1 could function as a tumor suppressor in a number of cancers including colorectal, ovarian and breast cancer ,28,29. HWhat’s more, the enforced expression of LINC00491 dramatically reduced the MTSS1 protein expression, while it was reversed following treatment with MG132 A. These The ubiquitin-proteasome pathway is a key posttranslational modification resulting in the degradation of proteins. To explore whether LINC00491 decreases the MTSS1 protein level through ubiquitin–proteasomal degradation, denature-IP experiments were carried out to examine the ubiquitination level of MTSS1 when LINC00491 was knocked down or not in A549 and H1299 cell lines B. The siYang et al. revealed the activation of Wnt/β-catenin-signaling attributes in tumor growth and metastasis in lung cancer . PreviouWe next further investigate whether LINC00491 interacts with MTSS1 to promote the malignance of the LUAD cell. Colony formation and EdU assays showed that the overexpression of MTSS1 dramatically attenuated the proliferation capability promoted by LINC00491’s overexpression . TransweAdditionally, for in vivo experiments, the xenograft tumor model verified that the overexpression of MTSS1 reversed the proliferation ability of the LUAD cell in vivo, enhanced by LINC00491’s overexpression E. The IHTo explore the clinical significance of the LINC00491–MTSS1 complex in LUAD, the levels of LINC00491 and MTSS1 were measured in LUAD samples. As shown in Recent studies have revealed that lncRNAs are aberrantly expressed between diverse tumor types and normal tissues, which suggests their potential value in cancer diagnosis, monitoring, prognosis or response to molecular therapy. Lung adenocarcinoma (LUAD) is the major histopathological subtype of lung cancer, occupying 60% of NSCLC, and more than 40% of lung cancer . DespitePrevious studies on lncRNAs showed that they are involved in various biological functions through different molecular mechanisms. Some lncRNAs link themselves to the transcription site to regulate the expression of a gene via interaction with proteins ,37. SomePrevious studies reported MTSS1 as a tumor and/or metastasis suppressor whose expression is negatively correlated with poor prognosis in some solid tumors, while the specific mechanism of low MTSS1 expression in LUAD is little reported . Here, wAlthough our findings illuminate that LINC00491 activates Wnt/β-catenin signaling via promoting MTSS1 ubiquitin–proteasome degradation, it is currently not clear how LINC00491 expression increases in the LUAD. Some microRNAs were reported to regulate MTSS1 in other cancer types such as miR-96-5p in ovarian cancer and miR-23a in colorectal cancer ,42. In tIn summary, our study revealed that LINC00491 expression is upregulated in LUAD and that high expression of LINC00491 is positively correlated with tumor metastasis and poor survival. LINC00491 interacts with MTSS1 directly to facilitate ubiquitination degradation. Consistently with previous findings that MTSS1 is a tumor/metastasis suppressor, our study reveals that the LINC00491/MTSS1 axis facilitates tumor malignant progression via activating Wnt/β-catenin signaling. This study further confirms the diversity and complexity of lncRNA-protein interactions, and suggests that the LINC00491/MTSS1 axis could be a potential target in LUAD diagnosis and therapeutic strategies.


P < 0.001) groups. CSF GAP-43 correlated with CSF phosphorylated tau 181(p-tau) , and had high diagnostic accuracy in differentiating tau positive status vs. tau negative status . CSF GAP-43 was particularly elevated among individuals with tau positive status. High CSF GAP-43 was associated with longitudinal deterioration of cognitive scores and brain neuroimaging findings. CSF GAP-43 was associated with a clinical diagnosis of AD dementia and with an individual’s tau status, cognitive measurements and findings from neuroimaging. This study implies that CSF GAP-43 as a biomarker of synaptic dysfunction could predict the disease progression of AD patients.To test whether cerebrospinal fluid (CSF) growth-associated protein 43 (GAP-43) concentration is elevated in Alzheimer’s disease (AD) dementia and its associations with other hallmarks of AD, we examined the CSF GAP-43 measurements of 787 participants , 415 individuals with mild cognitive impairment (MCI) and 127 individuals with AD dementia) from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) study. Associations were investigated between CSF GAP-43 and clinical diagnosis, Aβ/tau/neurodegeneration (AT(N)) status, CSF and blood biomarkers of AD, cognitive measurements and brain neuroimaging findings. CSF GAP-43 levels were increased in patients with AD dementia  compared with the CN  and MCI  ( Synapses are important for cognitive function, and synaptic impairment is one of the pathologic features of AD. A quantitative morphometric study has demonstrated a 25–35% decline of synaptic density in temporal and frontal cortical biopsies of patients (2–4 years) into the onset of AD2. In AD patients the synaptic loss primarily occurs in the neocortex and hippocampus3. Synaptic damage and cell loss leads to brain atrophy, and synaptic dysfunction is associated with cognitive impairment in AD4. Therefore, evaluating synaptic dysfunction in vivo would guide AD clinical research and could provide biomarkers for outcome assessment in AD clinical trials. In recent years, progress has been achieved to evaluate synaptic biomarkers in the cerebrospinal fluid (CSF). Depending on the localization of the synaptic protein, synaptic biomarkers can be divided into pre- and postsynaptic biomarkers. Several studies reported CSF concentrations of the presynaptic proteins, such as growth-associated protein 43 (GAP-43), synaptosomal-associated protein 25 (SNAP-25) and synaptotagmin-1, as well as postsynaptic protein neurogranin are altered in AD patients8. Among those synaptic proteins, GAP-43 plays an important role in the learning and memory storage process10.Worldwide, Alzheimer’s Disease (AD) is one of the most common types of neurodegenerative disorders and is the leading cause of dementia. The neuropathological features of AD include extra cellular aggregation of amyloid β (Aβ) plaques, intracellular deposition of neurofibrillary tangles containing phosphorylated tau protein and synaptic loss11. GAP-43 is phosphorylated by protein kinase C and then interacts with other proteins to facilitate axonal outgrowth and vesicular cycling12. In cultured hippocampus neurons, GAP-43 colocalizes with axonal marker tau protein13. GAP-43 is involved in axonal outgrowth, neuroplasticity and memory formation16. In the central nervous system, levels of GAP-43 protein are high in the cerebellum, neocortex, entorhinal cortex, hippocampus, olfactory bulb and retinal cells17. As a biomarker of synaptic dysfunction, previous studies reported that the concentration of CSF GAP-43 was elevated in AD patients20, and in this present study we analyzed whether CSF GAP-43 is associated with other CSF and blood biomarkers in AD and whether the baseline levels of CSF GAP-43 could predict changes in cognitive measurements and neuroimaging findings over time.In human, GAP-43 is a presynaptic protein consisting of 238 amino acids, and is located on the cytoplasmic side of the presynaptic membranehttp://adni.loni.usc.edu/) in July 2021. ADNI is a public–private project launched in 2003 and led by principal investigator Michael W. Weiner, MD. The primary goal of the ADNI project is to develop clinical, neuroimaging, genetic and bio fluid biomarkers for the early diagnosis and monitoring of AD progression. Regional ethical committees of all participating institutions approved the ADNI study, and all methods were carried out in accordance with relevant guidelines and regulations. Written informed consent was obtained from all study participants.Data used in this study were downloaded from the Alzheimer’s disease Neuroimaging Initiative (ADNI) database  controls, MCI and AD dementia were based on cognitive assessments. CN participants had a Mini-Mental State Examination (MMSE) score of 24 or higher and a Clinical Dementia Rating Scale (CDR) score of 0. Participants with MCI had an MMSE score of 24 or higher and CDR score of 0.5, objective memory loss was determined by delayed recall of the Wechsler Memory Scale Logical Memory 2, preserved activities of daily living, and absence of dementia. Participants with AD dementia had MMSE scores between 20 and 26 and CDR scores ranging from 0.5 to 1, fulfilling the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer Disease and Related Disorders Association criteria for probable AD22.The inclusion and exclusion criteria for ADNI participants have been described previously5.CSF GAP-43 levels were measured by an in-house enzyme-linked immunosorbent assay (ELISA) method developed at the Clinical Neurochemistry Lab, University of Gothenburg, Sweden. ELISA was developed by using a combination of monoclonal GAP-43 antibody NM4 (coating antibody) and polyclonal GAP-43 antibody ABB-135 (detector antibody) to recognize the C-terminal of GAP-43. The ELISA assay range of CSF-GAP43 was from 312 to 20,000 pg/ml. Quality control CSF samples were from the Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden. Details of the ELISA assay of CSF GAP-43 has been described previously24. CSF Aβ42, CSF p-tau and CSF t-tau were utilized to define amyloid pathology (A), tau pathology (T) and neurodegeneration (N) respectively according to the ATN framework25. The published cutoff values 26 were used to define the positivity of A/T/N status.CSF samples were collected by lumbar punctures from a subset of participants, and levels of CSF Aβ42, CSF total tau (t-tau), and CSF phosphorylated tau 181 (p-tau) were analyzed by electrochemiluminescence immunoassays (ECLIA) on a fully automated Elecsys cobas e 601 instrument as previously described27. Both plasma p-tau181 and neurofilament light (NfL) levels were analyzed by the Single Molecule array (Simoa) technique at the Clinical Neurochemistry Lab, University of Gothenburg, Sweden. Plasma p-tau181 was measured by an in-house assay, using a combination of two monoclonal antibodies (Tau12 and AT270) and targeted N-terminal to mid-domain forms of p-tau18128. Plasma NfL levels were analyzed by the Simoa technique using a combination of monoclonal antibodies, and purified bovine NfL was used as a calibrator for the measurements29.Plasma Aβ42 and Aβ40 levels were measured at Bateman lab, Washington University School of Medicine. Immunoprecipitation of targeted Aβ isoforms was performed in the KingFisher (Thermo) automated immunoprecipitation platform by using an anti- Aβ mid-domain antibody (HJ5.1), and subsequently digested with Lys-N protease then analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS) as previously described30. Volumetric data of the medial temporal lobe, and hippocampus were used in our analysis and were adjusted for intracranial volume. Positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) image data was used to identify hypometabolic regions related to pathological metabolic change in MCI and AD, and FDG-PET data was processed by Helen Wills Neuroscience Institute, University of California Berkeley and Lawrence Berkeley National Laboratory. The mean counts from composite metaROI (region of interest) of left angular gyrus, right angular gyrus, bilateral posterior cingulate, left inferior temporal gyrus, right inferior temporal gyrus relative to pons and cerebellar vermis reference region were adopted for FDG-PET assessment31.Structural brain imaging was performed by 3.0 T MRI scanners, T1 weighted images in NiFTI format were quantified by FreeSurfer version 5.1 for regional volumes according to the 2010 Desikan-Killany atlasThe cognition level of participants in the cohort was evaluated by the Mini-Mental State Examination (MMSE), the 11 item version of the Alzheimer Disease Assessment Scale Cognitive Subscale (ADAS-COG 11) and the CDR Scale Sum of Boxes (CDR-SB).P values < 0.05 were considered statistically significant, and Stata version 16  statistical software was used for all statistical analyses.Descriptive statistics between diagnostic groups were compared using ANOVA, Kruskal–Wallis, chi-square test according to the distribution of each variable. CSF GAP-43 data had a skewed distribution  and were log transformed to produce a normally distributed dataset. The differences of log transformed CSF GAP-43 between tau negative and tau positive groups were examined by the student’s t test, differences between multiple groups were compared with one-way analysis of variance (ANOVA) and the Tukey post hoc test. Receiver operating characteristic (ROC) analyses were used to evaluate the diagnostic accuracy of tau status by CSF GAP-43, and DeLong test was performed to compare area under the curve (AUC) values. Marginsplot was created using Stata software to predict the probability of tau positive status based on CSF GAP-43 levels. Decision curve analysis was performed using the DCA function code in Stata software. Linear regression models were used to test associations between biofluid markers and CSF GAP-43. Linear mixed-effect models were used to test associations between CSF GAP-43 with longitudinal cognition scores, and imaging of brain structures. These models included the interaction between time and CSF GAP-43 as a predictor, and random slope and random intercept for time and unstructured covariance structure for random effects. All outcome variables in linear mixed-effect models were standardized to z scores so that the effects could be directly compared between associations. All regression analyses were adjusted for age, sex, years of education and APOE ε4 status, intracranial volume was also adjusted for in models involving imaging of brain structures. Two-sided https://adni.loni.usc.edu/wp-content/uploads/how_to_apply/ADNI_Acknowledgement_List.pdf).ADNI was approved by the regional ethical committees of all participating institutions (A complete listing of ADNI participating institutions can be found at: P < 0.001).In this study we included 787 participants with available CSF GAP-43 measurements, the mean (SD) age was 72.35 (7.3) years old, and 416 (52.86%) were men. The mean (SD) education years of the participants was 16.27 (2.60) years, among them 430 (54.64%) were APOE ε4 allele negative , the A+T–N– group (P < 0.001) and the A–T–N– group (P < 0.001)  and the A–T–N– group (P = 0.001), the difference between the A+T–N– group and the A–T–N– group was not statistically significant , tau positive individuals with MCI had higher CSF GAP-43 levels than tau negative individuals with MCI (P < 0.001), and CSF GAP-43 levels were higher in tau positive controls compared with tau negative controls (P < 0.001)     Fig. A. We fur01) Fig. . ROC ana03) Fig. B. To est03) Fig. C. Decisi03) Fig. D.Figure We further classified the participants based on the presence of amyloid pathology (A) and tau pathology (T) in CSF into three groups: A–T–, A+T–, A+T+. CSF GAP-43 levels were higher in the A+T+ group compared with the A–T– and A+T– group, there were no statistically significant differences of CSF GAP-43 levels between the A–T– and A+T-groups  this would put the individual at a place on the AD continuum called AD pathological change. If both Aβ and tau abnormalities were present, but neurodegeneration was absent (A+T+N–), the individuals could be considered as having AD even without exhibiting signs of neurodegeneration. If all three domains of Aβ, tau and neurodegeneration were present (A+T+N+), this would reflect a more advanced stage on the AD continuum compared to an A+T+N– profile. In our study, we demonstrated that CSF GAP-43 levels increased along the AD continuum. Besides, CSF GAP-43 levels were also associated with cognitive deficits and neuroimaging finds both at baseline and during longitudinal follow up.The present study showed that CSF GAP-43 was elevated in the clinical AD dementia group and in tau positive individuals. CSF GAP-43 was correlated with CSF p-tau, CSF t-tau and plasma p-tau. CSF GAP-43 levels were much higher in the tau positive group compared to the tau negative group, which distinguished tau positive status from tau negative status. The 2018 AD research framework proposed a biomarker classification system called the Aβ/tau/neurodegeneration (AT(N)) system, which defined AD as an AD continuum based on core neuropathological changes in vivo32. The significant role of synaptic dysfunction in the pathology of AD promotes the analysis and quantification of synaptic proteins. GAP-43 is a synaptic membrane protein which plays an important role in the regulation of synaptic plasticity, learning and memory functionality16. Previous studies reported that the concentration of CSF GAP-43 was increased in AD20, and our results are in line with these reports. Our study demonstrated that CSF GAP-43 was correlated with CSF p-tau, CSF t-tau and plasma p-tau, which might reflect a common pathogenic process between GAP-43 and tau pathology. Our study also demonstrated that CSF GAP-43 could efficiently discriminate tau pathology status. A+T– represented a stage of amyloid pathological change and A+T+ indicated an advanced stage with both amyloid and tau pathologies present. CSF GAP-43 was particularly pronounced in A+T+ compared to A+T– individuals, thus CSF GAP-43 could efficiently discriminate between A+T– and A+T+ stages. It has been suggested that high concentrations of CSF t-tau represent axonal degeneration and high concentrations CSF p-tau represent the increased formation of neurofibrillary tangles, and that these two events are associated34. As CSF GAP-43 was highly correlated with CSF p-tau and CSF t-tau, this may indicate that increased CSF GAP-43 concentration is associated with the degeneration of axons or presynaptic terminals, or the regeneration of axons and/or synapses35.There is much evidence that synaptic loss is correlated with cognitive decline in AD, and synaptic dysfunction is one of the earliest detectable changes in many neurodegenerative diseases, which may appear even before neuronal loss36. A previous study utilized synaptosomes from the cortex of postmortem human subjects and transgenic rat models of AD to elucidate the time sequence of Aβ and tau pathology in synaptic terminals. This study demonstrated that Aβ accumulated in synaptic terminals in the early stages of AD, and these changes appeared before the accumulation of synaptic p-tau, the accumulation of p-tau in synaptic terminals occurred in the late stages of AD37. Aβ initiated synaptic dysfunction via tau pathology, and without the presence of tau pathology there would not be resulting synaptic dysfunction and memory impairment38. In our study we showed that CSF GAP-43 were correlated with CSF p-tau and plasma p-tau, and there was no correlation between CSF GAP-43 and CSF Aβ42. This could be partially explained by CSF GAP-43 being a biomarker of synaptic dysfunction, and the accumulation of tau pathology being correlated with synaptic dysfunction and memory impairment. The accumulation of Aβ42 initiated all of these events.According to the amyloid cascade hypothesis, the soluble oligomer Aβ initiates tau pathology, and tau pathology leads to neuronal dysfunction and cell loss39. Tauopathies include but are not limited to AD, progressive supranuclear palsy (PSP), corticobasal syndrome (CBS) and some types of frontotemporal lobar degeneration (FTD), and pathogenic tau aggregation has different characteristics among these disorders40. A study of 662 individuals found that CSF GAP-43 concentration was increased significantly in clinical AD and CBS patients, while there were no concentration differences between both the control and clinical PSP groups, and behavioral variant FTD (bvFTD), and amyotrophic lateral sclerosis (ALS) with FTD groups5. The differences in the structural characteristics of tau aggregation and cellular localization among those disordered might lead to heterogeneous concentrations of CSF GAP-43 in comparison to the control group.Aβ pathology and tau pathology are two major pathological hallmarks of AD, and tau aggregation is the primary pathological feature of a category of clinically heterogeneous neurodegenerative disorders termed tauopathies2. Synaptic dysfunction has been associated with cognitive impairment in AD4. Our study showed that on neuroimaging, high CSF GAP-43 levels were associated with smaller hippocampus and medial temporal lobe volume, lower FDG-PET values at baseline, and accelerated reduction in hippocampus and medial temporal volume, greater decline of FDG-PET values over time. A neuroimaging study reported that synaptic density in the hippocampus area was decreased in AD patients41. Synaptic activity could be measured by cerebral glucose metabolism, and FDG-PET measures low cerebral glucose metabolism in regions of interest in AD patients, which associated with concurrent cognitive decline31.In this study we also found that CSF GAP-43 was associated with several cognitive and neuroimaging hallmarks of AD at baseline and over time. Specifically, high CSF GAP-43 levels were associated with MMSE, ADAS-COG 11, and CDR-SB scores at baseline as well as with accelerated deterioration of those cognitive measures longitudinally. Synapses are important for cognitive function, and synaptic loss is one of the pathologic features of AD42. Our study also lacks analysis of the longitudinal changes of CSF GAP-43 levels in AD, and longitudinal changes of CSF GAP-43 levels would better depict the features of CSF GAP-43 in AD. Finally, in this study due to lack of other synaptic biomarker’s measurements data , we only analyzed CSF GAP-43’s association with other core AD fluid biomarkers.The present study is limited by lacking data from other neurodegenerative disorders, which prevents our ability to test the disease specificity of CSF GAP-43 for AD, and whether CSF GAP-43 is associated with other neuropathological biomarkers such as α-synuclein, TDP-43. Another limitation of this study is its lack of evaluation of other tauopathies such as FTD, PSP, and CBS, which limits analysis of association between CSF GAP-43 and tau pathology in other tauopathies. For tau pathology assessment, in this study due to lack of tau PET finings, we used p-tau in CSF to quantify tau pathology, which was associated with CSF GAP-43 levels. However, previous studies have demonstrated that findings of tau PET neuroimaging were more closely associated with neurodegeneration than CSF tauIn conclusion, the CSF concentration of synaptic membrane protein GAP-43 was specifically elevated in tau positive individuals and could be used a biomarker for synaptic dysfunction. In addition to Aβ/tau/neurodegeneration (AT(N)) neuropathological biomarkers, CSF GAP-43 could be included as another neuropathological measurement of synaptic dysfunction in AD. In clinical study scenarios of AD, CSF GAP-43 could be used as another outcome measurement to predict longitudinal disease progression.Supplementary Information.


Morona Santiago and Los Rios provinces. In conclusion, the results highlight the complexity of the CSF control programs, the importance to improve the overall surveillance system and the need to inform decision-makers and stakeholders.Classical swine fever (CSF) is one of the most important re-emergent swine diseases worldwide. Despite concerted control efforts in the Andean countries, the disease remains endemic in several areas, limiting production and trade opportunities. In this study, we aimed to determine the risk factors and spatiotemporal implications associated with CSF in Ecuador. We analysed passive surveillance and vaccination campaign datasets from 2014 to 2020; Then, we structured a herd-level case–control study using a logistic and spatiotemporal Bayesian model. The results showed that the risk factors that increased the odds of CSF occurrence were the following: swill feeding (OR 8.53), time until notification (OR 2.44), introduction of new pigs during last month (OR 2.01) and lack of vaccination against CSF (OR 1.82). The spatiotemporal model showed that vaccination reduces the risk by 33%. According to the priority index, the intervention should focus on  The overall importance of pig production is related not only to meat consumption but also to cultural traditions. In Andean communities, pigs play a central role as a source of protein, festivities and savings [With the growing demand for animal protein in the Andean region, especially pork, diseases such as classical swine fever (CSF) are gaining importance as they are limiting local production and potential export opportunities for affected countries. The per capita consumption of pork has increased in Ecuador from 6.88 kg in 2009 to 10.9 kg in 2018 (Flaviviridae [Suidae family). Clinical signs are variable and depend on the viral strain, host immune response, age, general health status and concomitant infections [CSF is considered a relevant re-emerging viral disease of pigs, caused by Pestivirus of the family iviridae . The onlfections .The course of the disease includes acute, chronic and persistent forms according to their duration ,5. TransIn South America, the disease is considered endemic in Guyana, Suriname, the North and Northeast regions of Brazil and the Andean Community, and these regions struggle to implement successful control programs ,12.https://wahis.oie.int, accessed on 1 December 2022). In this regard, the NVS planned to enhance their analysis capacity and apply risk-based surveillance. One of the main challenges in applying risk-based surveillance is to identify the factors associated with the occurrence of CSF [The CSF eradication Project in Ecuador started in 2012; the first national vaccination campaign was gradually launched in 2014 with a locally produced lapinised Chinese vaccine strain . The hige of CSF ,17,18,19e of CSF , and PerFor Ecuador despite the importance and need for local CSF risk factors, little is still known concerning control measures and public policy. This is the first time that official data have been analysed in this regard. The objectives of this study were to determine the risk factors associated with the occurrence of CSF and to analyse the spatiotemporal implications in order to identify the most at risk locations.https://sistemas.agrocalidad.gob.ec/sizse/, accessed on 1 January 2021); (2) the Unified information manager (GUIA) developed by the NVS to manage cadastre, mass vaccination campaigns against CSF, and movements since 2016 . Shapefiles of administrative units of Ecuador were downloaded from the Institute of Statistics and Census (INEC) . Data were collected by the NVS from January 2014 to November 2020 in mainland Ecuador, excluding the Galapagos Islands, as they are a recognised CSF-free zone [https://CRAN.R-project.org/, accessed on 1 January 2022).All raw data were then imported and processed with R version 4.2.1  [The variables included in the analysis were selected based on biological plausibility, published literature, and considering their association with the occurrence of classical swine fever. ,23,24. Sch 2021)  and grou® CSFV), based on the double antibody sandwich (DAS) principle with a sensitivity of 97% and a specificity of 99% [® reagents [Laboratory testing was performed at the National Reference Laboratory (headquarters in Quito). Virus detection was carried out by a commercially available antigen ELISA screening test  animals with clinical signs consistent with CSF , or (b) An epidemiological link to CSFV. Confirmed positive cases were identified by the detection of antigens of CSFV ; the others with negative test results and lack of clinical signs were classified as controls . The passive surveillance system used a questionnaire  designed to obtain information provided by the owner of the animals and then registered online by the official veterinary; the information includes: demographic data of the owners and premises, geographic coordinates, chronology (dates of notification and follow-up), animal species, vaccination declaration, clinical signs, presumptive syndrome, collection of material, characteristics of samples, laboratory tests, animal population, animal movement and probable origin of the disease.The information was collected by trained NVS veterinarians following the data protection procedures of Ecuadorian authorities. The information recorded throughout the country was continually monitored by the national surveillance team (headquarters), who checked the data for completeness and errors. Additional information and descriptions of the questionnaire is available on All analyses were performed at the herd level and stratified according to CSF status (case or control). Variables were organised by type; continuous variables were transformed into dummies, setting their levels according to biological or legal cut-off points . Descripβi , assessing them at each step. Collinearity was analysed using variance inflation factors analysis [p > 0.05) [A multivariate logistic regression model was implemented to assess the association of explanatory variables with the outcome, using a manual forward stepwise selection . We inclanalysis . The gooanalysis , Receive > 0.05) ; Additio2, average temperature (°C) and average precipitation (mm), constructing several models. Temperature and precipitation were extracted from  at a spatial resolution of 2.5 arc-minutes (~5 km2). The analysis used the population and cases were restricted to 2017–2020 due to the lack of cadastral and vaccination information prior to the implementation of the official vaccination in 2017. Data were organised to contain the aggregated annual population over each parish (1040) using time-series missing value imputation  for areaYij observed in area i and time period j, modelled as:i) and time period (t) (Equation (1)). Three sets of components for ui is the correlated heterogeneity, which models the spatial dependence between the relative risks, and vi is the unstructured exchangeable component that models uncorrelated noise (Equation (2)).Ai represents the spatial group, Bj is the temporal group, and Cij is the space–time interaction group (Ai = ui + vi) using the most popular model to spatially define disease, also known as Besag–York–Mollié (BYM) [ui is modelled with the conditional autoregressive distribution (CAR) [Bj = βtj). Using an independent and identically distributed Gaussian random effect (iid). (Cij = δitj), where ui + vi is an area random effect, βtj is a linear trend term in time tj, and δitj is an interaction random effect between area and time (Equation (3)) [Parish vaccination coverage was adjusted considering the population and the doses applied against CSF, considering 1.55 as the average number of doses a pig receives in a calendar year, according to the average lifespan from birth to slaughter (234 days) . We usedié (BYM) , where ton (CAR) , smoothiion (3)) . p value. To suggest a parish that required a priority of care, we used the priority index (PI) which is a risk-based percentage scale that ranks the units of analysis, given by the fitted effects weighted by their probability and a cut-off value [To evaluate the models, we used the deviance information criterion (DIC) and the posterior predictive ff value . The modff value . We usedhttps://cran.r-project.org/, accessed on 1 May 2022), and the following packages were used: Tydiverse [All analyses were run in R V4.2.0 , herd level (n = 9) and animal level (n = 2). Time until notification was transformed considering a cut point of 7 days (one week) since the onset of clinical signs. The pigs’ population on the premise was transformed, considering cut points of 25 and 190 pigs, and considering percentiles 75 and 95 of the national population due to previous national limits for backyard and commercial premises. The age of the animals considered cut-off points of 2 and 6 months due to the official CSF vaccination recommendation: first dose is applied after 45 days and revaccination occurs at 180 days of age  for cases and one week (7.0 days) for controls. The median premise population was similar for cases (13.5 pigs) and controls. The mean age of pigs was similar for cases and controls (~5 months) .Farmer vaccination declaration was higher for case herds (71%) than for control herds (6%). Recording of vaccination  was lower for cases than for controls. Both cases and controls had a high percentage of swill feed use. The entry of animals within the last 30 days happened in 39% of cases and 22% of controls. Only 5% of the cases and 4% of controls have other species on the property .Historical case presentation decreased over the years, with the highest proportion of cases (48%) occurring between 2014 and 2015 and the lowest (14%) occurring between 2019 and 2020. The proportion of cases (43%) and controls (45%) was higher in the highlands. More than half of the case notifications (51%) were reported by the owner, followed by health sensors (42%), which are volunteers selected by the NVS to enhance the surveillance system directly from communities across the country. There was a higher proportion of cases in the third community network, known for their high density of backyard producers and indigenous communities , locatedThe surveillance database contained 1254 questionnaires, 338 of which were confirmed CSF cases. The premise categories were 50.79% family (637), followed by 28.39% commercial (356), 17.98% backyard (224) and 0.03% industrial (37); the distribution of cases and controls over time is illustrated in Distributed by months, the mean number of cases was 4.39 ± 3.09; with controls, the mean was 11.31 ± 6.38 There was a tendency to increase twice a year around April and October .p < 0.20). We found a paradoxical fit (Type III error) opposite of the true effect [We ran 15 univariable models; twelve of the assessed variables were associated with CSF  had an incorrect direction of association (self-declaration of vaccination) or that were at the animal level  were excluded. Variables that showed a strong association (p < 0.0001) in the univariable model maintained their individual and model significance when adjusted in the final multivariable model ; also, there was no correlation between residuals. Details of the manual stepwise model construction, analysis and the predicted probability map of occurrence aggregated by parishes is available in Factors that substantially increased the odds of CSF occurrence at the herd level were swill feeding (OR 8.53), time until notification (OR 2.44), entry of animals in the last 30 days (OR 2.01) and lack of CSF vaccination (OR 1.82) . The finp = 0.55). The final neighbour list contained 1006 parishes with an average of 5.71 parish neighbours. The average parish area was 198.03 ± 249.48 km2, with a range from 2.23 to 2429.64 km2. The annual average of registered premises was 115,411.8, housing an average of 1,633,922 pigs. The Ecuadorian administrative division has 1040 parishes; 16 were removed because of them being islands and 18 were located in the Amazon rainforest with no record of domestic pigs. The length of the neighbouring areas was 4024 (1006 for each year), with 271 (6.49%) imputed gaps. When comparing the imputed data with the original dataset, they were not significantly different ; the highest average vaccination coverage was 81% in 2019. The number of applied doses increased from 1.8 million in 2017 to 2.4 million in 2020. The annual average of the premises was 115,411 .What stands out in Temperature and precipitation in Ecuador are modulated by the Andes mountains, warmer temperatures in the Amazon and Coastal regions and cooler temperatures in the highlands. There is a range difference of 11 °C in the coastal regions, 20.5 °C in the highlands and 18.9 °C in the Amazon. Precipitation on the Amazon is almost three times higher than the highlands and two times compared with the Coastal .2, they behaved as an expected protective factor, which means that an increase in one SD in the doses applied per kilometre decreased the risk by 33%. Temperature was a risk factor, considering that an increase in one SD in temperature degree increased the risk by 16.7%. Precipitation had no effect: RR = 1.00 (1.00–1.001)  .The proportion of variance explained by each component was 57% for the random effect (iid), a major contributor to the explained variance, and 43% for the spatiotemporal (besag). The DIC mean deviance was 1140 and the effective number of parameters was 136.7. The spatial distribution of risk at parish scale is shown in Tundayme located in the eastern Amazon, followed by Tachina in the north-western and Paletilla in the southern zone (Morona Santiago (3.68), Los Rios (3.12) and Santa Elena, (3.07); the average relative risk intended to prioritise prevention and control activities by province is available in According to the priority index (PI), primary parishes of concern include ern zone . The proWhen countries mount resource-intensive control strategies for a high-impact disease such as CSF but fail to reach the goal of control and elimination, a deeper analysis of the disease dynamics and the implemented control interventions is needed to identify strategic intervention points. Despite the fact that, in general terms, many CSF risk factors are known, their relevance in the specific setting of a pig sector and the respective control program is ideally assessed using all available data.Swill feeding is one of the main risk factors for CSF transmission; it is common and rooted in the cultural tradition of backyard producers . TherefoVaccination misreporting could be related to the producer’s lack of knowledge regarding veterinary treatments linked with injections . Fear associated with owners’ legal responsibilities and misunderstandings during the interviews may also lead to misreporting . In IndoThe increased risk of pigs at the age of 3–6 months (univariate model) could be related to the fact that young animals may be more exposed to CSFV because this is the age at which, in rural communities, they are moved to animal markets, using various means of transport. Additionally, this reflects a complicated age from the immunological perspective because maternal immunity fades out after three months , and aniMaternal-derived antibody (MDA) interference is the most common factor affecting the induction of protective immunity against CSFV ; in ThaiSome cases occurred on premises with previous vaccination records, possibly related to illegal movements of unvaccinated animals  or vacciThe identified individual parish risk could help identify neglected territories; as in many developing countries with limited resources for disease control, prioritisation is often done on the basis of historic surveillance information. Therefore, reduced surveillance sensitivity may leave areas of high risk unnoticed. Our spatial model included all parishes and considered the influence of their neighbours to improve the predictions . ConceptThe identification of the risk factors should respond to the initial demand of the NVS and contribute to the implementation of a risk-based surveillance strategy for animal diseases. As risk factors are specific for each disease, new studies could be implemented using depurated data and methodology for prevalent diseases where their symptomatology could be confused with CSF, such as the porcine reproductive and respiratory syndrome ,64, as wThe purpose of the current study was to determine the risk factors and spatiotemporal implications associated with CSF in Ecuador. The results show once again the complexity that the CSF control program is facing, particularly if the pig sector is diverse and comprises a large share of premises falling under the backyard category. Here, NVS faces risky production methods combined with a reduced knowledge of disease prevention and compliance with sanitary regulations.One of the more significant findings from this study is the identification of swill feeding, time until notification, introduction of new pigs and the lack of vaccination, as the risk factors; the second major finding is the parish priority index based on spatiotemporal risk. We hope these results would be useful to improve intervention focusing on specific parishes countrywide.The results highlight the importance of improving the overall surveillance system and the need for better methods to inform decision-makers and stakeholders.


We reviewed 51 studies published through December 2021 reporting metagenomic next generation sequencing (mNGS) directly applied to the cerebrospinal fluid (CSF). This approach, potentially circumventing the above-mentioned limitations, indicated 1,248 investigated patients, and 617 patients dually investigated by routine diagnosis and mNGS, in whom 116 microbes were detected, including 50 by mNGS only, nine by routine methods only, and 57 by both routine methods and mNGS. Of 217 discordant CSF findings, 103 CSF samples were documented by mNGS only, 87 CSF samples by routine methods only, and 27 CSF samples in which the pathogen identified by mNGS was different than that found using routine methods. Overall, mNGS allowed for diagnosis and genomic surveillance of CAM causative pathogens in real-time, with a cost which is competitive with current routine multiplex RT-PCR. mNGS could be implemented at point-of-care (POC) laboratories as a part of routine investigations to improve the diagnosis and molecular epidemiology of CAM, particularly in the event of failure of routine assays.Current routine diagnosis of community-acquired meningitis (CAM) by multiplex real-time polymerase chain reaction (RT-PCR) is limited in the number of tested pathogens and their full characterisation, requiring additional  Cryptococcus in HIV-infected patients, in whom Cryptococcus most frequently causes fungal meningitis, with 223,000 new cases each year and 81% mortality (Acanthamoeba (Community-acquired meningitis (CAM), a life-threatening condition with 40% mortality , warrantortality  and amoethamoeba . The preMetagenomic next generation sequencing (mNGS) directly applied to cerebrospinal fluid (CSF) emerged less than 10 years ago, in the specific context of healthcare-associated meningitis , as an aThis study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . A systeStudies with the following criteria were included in this review: Studies using mNGS-CSF for: (1) case reports; (2) prospective series; (3) retrospective series for non-routinely detected pathogens” . Review Bibliographic searches identified 3,341 articles and, following the Prisma diagram , 2,080 aIn 2015, the first reported application of mNGS directly on CSF (and brain biopsy) enabled the post-mortem diagnosis of Astrovirus-related encephalitis in an immunocompromised patient . A 7-yeag), filtration and vortexing in 12 studies, DNase treatment in four studies  using Burrows-Wheeler Alignment (BWA) in 40% of studies. Non-mapped reads could be assembled using Spades and de novo assembly or directly against the reference genome using the BWA and CLC Genomic workbench  as the only amoeba detected in a CSF sample  in whom routine diagnostic methods completely missed N. fowleri  CSF samples, including no pathogen documentation in 566 CSF samples and concordant identification in 465 CSF samples. Discordant results were observed in 217 (17.4%) CSF samples, including 87 CSF samples documented by routine methods only, 103 CSF samples documented by mNGS only, and 27 CSF samples in which documented microorganisms differed from routine methods and mNGS. Altogether, the 116 detected microorganisms included 63 bacteria, 38 viruses, 10 fungi, and five parasites. mNGS detected 106/116 (91.4%) different microorganisms, including 50 (43.1%) detected by mNGS only, whereas 56 microorganisms were detected by both routine methods and mNGS. In addition, five microorganisms were detected by routine molecular diagnosis only, four microorganisms by culture only and one  culture . FurtherF sample . Of the onavirus , Coxsackps virus , Saint-Lis virus , Powassaan Virus , Toscanana virus , Jamestoon virus , Enterovon virus , and Hep E virus , while o E virus . Bacteriur cases , Ureaplaee cases  and one eumoniae , Pasteurultocida , Enterocfaecalis , Nocardiarcinica , Streptocus suis , and Psycter sp. . Toxopla fowleri , while Cningitis .A review of published data indicated that in 4/217 cases, mNGS failed to detect a pathogen when the pathogen had been detected by real-time polymerase chain reaction (RT-PCR) with a Ct > 37, mainly due to a low pathogen inoculum , as well1)Storage conditions: storage of the CSF at temperature above -80°C may cause nucleic acid molecule damage. Moreover, lack of proper storage in the presence of RNase inhibitors, will likely result in RNA viruses undergoing some degradation.2)Limited CSF volume affecting the DNA quantity used for NGS library preparation , especia3)3/mL) increasing the ratio of human genome, which required microbial genome enrichment   , meaning enzymes ,b, 2022.e agents . Moreovee agents ,c, with e agents , and obse agents . The sene agents ; 2021. Ie agents . Howevere agents .in silico antibiotic susceptibility testing was added, even in cases of uncultured bacteria.The mNGS approach is not currently standardised and remains in its infancy. Microbiologists may, therefore, wish to develop different strategies to investigate RNA and/or DNA pathogens, relying on available mNGS materials and reagents as well as mNGS platform, and in-house pipelines using local epidemiology-driven, specific pathogen genome databases . FinallyAs a time and cost-effective approach, real-time mNGS could be implemented in POC laboratories for the diagnosis of CAM, especially with regards to undocumented meningeal disease .MM: literature search, data collection, data cleaning, data interpretation, validation, and writing of the manuscript. JL and MD: design of the study, data interpretation, validation, funding, critical review of the manuscript, coordination, and direction of the work. The data presented here were extracted by MM and validated by JL and MD after review. All authors have read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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