The potential of historical spy-satellite imagery to support research in ecology and conservation.

Remote sensing data are important for assessing ecological change, but their value is often restricted by their limited temporal coverage. Major historical events that affected the environment, such as those associated with colonial history, World War II, or the Green Revolution are not captured by modern remote sensing. In the present article, we highlight the potential of globally available black-and-white satellite photographs to expand ecological and conservation assessments back to the 1960s and to illuminate ecological concepts such as shifting baselines, time-lag responses, and legacy effects. This historical satellite photography can be used to monitor ecosystem extent and structure, species' populations and habitats, and human pressures on the environment. Even though the data were declassified decades ago, their use in ecology and conservation remains limited. But recent advances in image processing and analysis can now unlock this research resource. We encourage the use of this opportunity to address important ecological and conservation questions.

Use of SPY angiography in tissue assessment for wound healing outcomes after breast reconstruction.

OBJECTIVE: The objective of this study was to evaluate whether a systematic image assessment protocol using SPY Elite images (LifeCell Corp., US) of viable tissue at the periphery of the surgical field was associated with positive wound healing outcomes following mastectomy and breast reconstruction. METHOD: Patients undergoing mastectomy and subsequent breast reconstruction surgery at a single tertiary medical centre were included. SPY images were prospectively analysed using a systematic image assessment protocol, and an absolute value of mean fluorescence was calculated by measuring peripheral, in-situ tissue from each image. Patient medical records were retrospectively reviewed for demographics, surgical characteristics and postoperative outcomes. These variables were statistically tested for associations with mean fluorescence. RESULTS: A total of 63 patients were included in the final analysis. We found that objectively determined mean fluorescence values were not statistically significantly associated with postoperative complications. CONCLUSION: In this study, objectively measured mean fluorescence values representing breast tissue remaining after dissection showed little utility in the assessment of postoperative wound healing outcomes as they did not identify patients who would later have complications of wound healing. DECLARATION OF INTEREST: The authors have no conflicts of interest to declare.

Direct-to-Implant Retropectoral Dual Plane Approach With Autologous Inferior-Based Dermal Flap: Does SPY-Elite Laser Angiographic System Reduce Complication Rates?

PURPOSE: The study aims to investigate the complications and long-term outcomes associated with retropectoral DTI breast reconstruction with IDF utilizing the SPY-Elite laser angiographic system. MATERIAL AND METHOD: This retrospective study was conducted from June 2017 to January 2023. We examined 52 patients (85 breasts) treated with a direct-to-implant retropectoral dual plane approach with IDF implant coverage. Informed consent was duly obtained from every participant. Inclusion criteria dictated that patients should have medium to large breasts and a second or third degree of ptosis, as per the Regnault ptosis scale. During the intraoperative evaluation, the mastectomy flaps and IDF were assessed with the SPY-Elite laser angiographic system using near-infrared imaging. We recorded patient demographics, characteristic data, and complications. RESULTS: A total of 52 patients, aged 27 to 63, underwent 85 mastectomies using a direct-to-implant retropectoral approach with inferior dermal flap. The average age of the patients was 48, and their average body mass index was 30.8, with a range of 28 to 43. The distance from the nipple to the inframammary fold varied between 14 and 24 cm. The implants used had an average size of 275 cc, ranging from 250 to 650 cc. Textured anatomic implants with either moderate plus or high profile were used in all cases. The sternal notch to nipple distance for these patients ranged from 24 to 38 cm. During the evaluation using the SPY-Elite laser angiographic system, insufficient distal marginal perfusion was detected in five out of 85 inferior dermal flaps, measuring between 2 and 5 cm2. These areas were subsequently debrided, and the reconstructions were successfully completed, representing 5.8% of cases. No instances of necrosis related to IDF have been observed. There have been no failed assessments conducted by SPY ICG. In total, the complication rate was 15.2%, with minor complications occurring in 8.2% of the breasts (7 out of 85) and major ones in 7% (6 out of 85). The subjects were monitored for an average of 14 months, the duration ranging from 12 to 24 months. CONCLUSION: Inferior dermal flaps have considerable advantages, such as a natural autologous blood supply, a more realistic tissue thickness and texture, lower costs, and better tolerance to post-reconstruction radiation. Moreover, using the IDF technique and assessing the perfusion of IDF and mastectomy flaps through the SPY-Elite laser angiographic system appears to be a dependable, efficient way to achieve good cosmetic results in one operation, eliminating the need for additional surgeries. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine Ratings, please refer to Table of Contents or online Instructions to Authors www.springer.com/00266.

Impact of spy fiction on cognition: a case study.

Reading spy fiction has imprinted its traces on the thinking patterns of the readers that encourage them to consider it as a real event in their life. This case study is about a 37 year old woman, referred by a senior clinical psychologist with complaints of having the feeling of being monitored through a device and hearing the voices of the people. The brief therapeutic plan was based on Cognitive Behavioural Therapy which focused on developing insight, identifying cognitive errors, and enhancing her socialisation skills. This case report holds its implications to drift the attention of the readers and clinicians toward the issue of consuming content that may potentially have an impact on its reader's mental health.

Association of baseline ROR1 and ROR2 gene expression with clinical outcomes in the I-SPY2 neoadjuvant breast cancer trial.

PURPOSE: ROR1 and ROR2 are Type 1 tyrosine kinase-like orphan receptors for Wnt5a that are associated with breast cancer progression. Experimental agents targeting ROR1 and ROR2 are in clinical trials. This study evaluated whether expression levels of ROR1 or ROR2 correlated with one another or with clinical outcomes. METHODS: We interrogated the clinical significance of high-level gene expression of ROR1 and/or ROR2 in the annotated transcriptome dataset from 989 patients with high-risk early breast cancer enrolled in one of nine completed/graduated/experimental and control arms in the neoadjuvant I-SPY2 clinical trial (NCT01042379). RESULTS: High ROR1 or high ROR2 was associated with breast cancer subtypes. High ROR1 was more prevalent among hormone receptor-negative and human epidermal growth factor receptor 2-negative (HR-HER2-) tumors and high ROR2 was less prevalent in this subtype. Although not associated with pathologic complete response, high ROR1 or high ROR2 each was associated with event-free survival (EFS) in distinct subtypes. High ROR1 associated with a worse EFS in HR + HER2- patients with high post-treatment residual cancer burden (RCB-II/III) (HR 1.41, 95% CI = 1.11-1.80) but not in patients with minimal post-treatment disease (RCB-0/I) (HR 1.85, 95% CI = 0.74-4.61). High ROR2 associated with an increased risk of relapse in patients with HER2 + disease and RCB-0/I (HR 3.46, 95% CI = 1.33-9.020) but not RCB-II/III (HR 1.07, 95% CI = 0.69-1.64). CONCLUSION: High ROR1 or high ROR2 distinctly identified subsets of breast cancer patients with adverse outcomes. Further studies are warranted to determine if high ROR1 or high ROR2 may identify high-risk populations for studies of targeted therapies.

Indocyanine Green Fluorescence Angiography in Minor Lower Extremity Amputations: A Useful Technique?

Complications remain despite conventional methods aimed at improving survivorship of lower extremity amputations. High rates of wound dehiscence, readmission, and revision surgery warrant the development of innovative methods to improve amputation survivorship. One such method employs the use of indocyanine green dye (ICG); an inert chemical injected intraoperatively which can be used to visualize dermal blood flow in real time. There is little objective data available to help guide the use of indocyanine green in limb salvage procedures. The present study compares a group of 31 patients undergoing minor lower extremity amputation with the use of indocyanine green with a control group of 62 patients in which traditional methods were used. Minimum follow-up of 9 months was obtained, leaving 93 patients in total for analysis. Success was defined as a healed amputation within 60 days of follow-up. Uneventful amputation healing occurred in 35.5% and 33.9% of indocyanine green and control patients, respectively. Overall, there was no significant difference in outcomes between groups (p = .965), or success versus failure (p = 1.0). Patient undergoing minor lower extremity amputation with the use of ICG fluorescence angiography did not have statistically inferior outcomes to patients who underwent amputations at the same level with traditional assessments of perfusion. Further research involving the use of this technique is warranted.

Role of Indocyanine Green Angiography in Free Flap Surgery: A Comparative Outcome Analysis of a Single-Center Large Series of 877 Consecutive Free Flaps.

Purpose This study aims to assess and validate the role and cost-effectiveness of indocyanine green angiography (ICGA) in free flap surgery outcomes. A new intraoperative protocol of whole-body surface warming (WBSW) for all free flap surgeries during the strategic "microbreaks" is also described. Methods A retrospective analysis of 877 consecutive free flaps, performed over 12 years, is presented. The results of the ICGA group ( n = 438) were compared with the historical No-ICGA group ( n = 439), and statistical significance was calculated for three crucial flap-related adverse outcomes and cost-effectiveness. ICGA was also used as a tool to show the effect of WBSW on free flaps. Results ICGA showed a notably strong statistical significance in decreasing two outcome parameters, namely, partial flap loss and re-exploration rate. It was also cost-effective. ICGA also demonstrated the positive role of WBSW in increasing flap perfusion. Conclusions Our study shows that the usage of ICGA for intraoperative assessment of flap perfusion can significantly reduce the partial flap loss and re-exploration rate in free flap surgeries in a cost-effective manner. A new protocol of WBSW is also described and recommended to increase flap perfusion in all free flap surgeries.

A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.

The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.

Using historical spy satellite photographs and recent remote sensing data to identify high-conservation-value forests.

High-conservation-value forests (HCVFs) are critically important for biodiversity and ecosystem service provisioning, but they face many threats. Where systematic HCVF inventories are missing, such as in parts of Eastern Europe, these forests remain largely unacknowledged and therefore often unprotected. We devised a novel, transferable approach for detecting HCVFs based on integrating historical spy satellite images, contemporary remote sensing data (Landsat), and information on current potential anthropogenic pressures (e.g., road infrastructure, population density, demand for fire wood, terrain). We applied the method to the Romanian Carpathians, for which we mapped forest continuity (1955-2019), canopy structural complexity, and anthropogenic pressures. We identified 738,000 ha of HCVF. More than half of this area was identified as susceptible to current anthropogenic pressures and lacked formal protection. By providing a framework for broad-scale HCVF monitoring, our approach facilitates integration of HCVF into forest conservation and management. This is urgently needed to achieve the goals of the European Union's Biodiversity Strategy to maintain valuable forest ecosystems.

Uso de Fotografías Históricas de Satélites Espía y Datos Recientes de Telemetría para Identificar Bosques de Alto Valor para la Conservación Resumen Los bosques de alto valor para la conservación (BAVC) tienen una importancia crítica para el suministro de servicios ambientales y biodiversidad pero enfrentan muchas amenazas. En donde hacen falta inventarios sistemáticos de los BAVC, como en partes del este de Europa, estos bosques siguen siendo ignorados y por lo tanto carecen de protección. Diseñamos una estrategia novedosa y transferible para la detección de BAVC con base en la integración de imágenes de satélites espía, datos contemporáneos de telemetría (Landsat) e información sobre las presiones antropogénicas actuales (p. ej.: infraestructura vial, densidad poblacional, demanda de leña, terreno). Aplicamos el método en los Cárpatos rumanos, para los cuales mapeamos la continuidad forestal (1955 - 2019), la complejidad estructural del dosel y las presiones antropogénicas. Identificamos 738,000 ha de BAVC. Más de la mitad de esta área fue identificada como susceptible a las actuales presiones antropogénicas y además carecía de protección formal. Mediante la aportación de un marco de trabajo para el monitoreo a escala amplia de los BAVC, nuestra estrategia facilita la integración de los BAVC dentro de la gestión y conservación de los bosques. Lo último es una necesidad urgente para alcanzar las metas de la Estrategia de Biodiversidad de la Unión Europea para mantener los ecosistemas boscosos valiosos.

Increased surface charge in the protein chaperone Spy enhances its anti-aggregation activity.

Chaperones are essential components of the protein homeostasis network. There is a growing interest in optimizing chaperone function, but exactly how to achieve this aim is unclear. Here, using a model chaperone, the bacterial protein Spy, we demonstrate that substitutions that alter the electrostatic potential of Spy's concave, client-binding surface enhance Spy's anti-aggregation activity. We show that this strategy is more efficient than one that enhances the hydrophobicity of Spy's surface. Our findings thus challenge the traditional notion that hydrophobic interactions are the major driving forces that guide chaperone-substrate binding. Kinetic data revealed that both charge- and hydrophobicity-enhanced Spy variants release clients more slowly, resulting in a greater "holdase" activity. However, increasing short-range hydrophobic interactions deleteriously affected Spy's ability to capture substrates, thus reducing its in vitro chaperone activity toward fast-aggregating substrates. Our strategy in chaperone surface engineering therefore sought to fine-tune the different molecular forces involved in chaperone-substrate interactions rather than focusing on enhancing hydrophobic interactions. These results improve our understanding of the mechanistic basis of chaperone-client interactions and illustrate how protein surface-based mutational strategies can facilitate the rational improvement of molecular chaperones.

Cold War spy satellite images reveal long-term declines of a philopatric keystone species in response to cropland expansion.

Agricultural expansion drives biodiversity loss globally, but impact assessments are biased towards recent time periods. This can lead to a gross underestimation of species declines in response to habitat loss, especially when species declines are gradual and occur over long time periods. Using Cold War spy satellite images (Corona), we show that a grassland keystone species, the bobak marmot (Marmota bobak), continues to respond to agricultural expansion that happened more than 50 years ago. Although burrow densities of the bobak marmot today are highest in croplands, densities declined most strongly in areas that were persistently used as croplands since the 1960s. This response to historical agricultural conversion spans roughly eight marmot generations and suggests the longest recorded response of a mammal species to agricultural expansion. We also found evidence for remarkable philopatry: nearly half of all burrows retained their exact location since the 1960s, and this was most pronounced in grasslands. Our results stress the need for farsighted decisions, because contemporary land management will affect biodiversity decades into the future. Finally, our work pioneers the use of Corona historical Cold War spy satellite imagery for ecology. This vastly underused global remote sensing resource provides a unique opportunity to expand the time horizon of broad-scale ecological studies.

Translational activation of maternally derived mRNAs in oocytes and early embryos and the role of embryonic poly(A) binding protein (EPAB).

Transcription ceases upon stimulation of oocyte maturation and gene expression during oocyte maturation, fertilization, and early cleavage relies on translational activation of maternally derived mRNAs. Two key mechanisms that mediate translation of mRNAs in oocytes have been described in detail: cytoplasmic polyadenylation-dependent and -independent. Both of these mechanisms utilize specific protein complexes that interact with cis-acting sequences located on 3'-untranslated region (3'-UTR), and both involve embryonic poly(A) binding protein (EPAB), the predominant poly(A) binding protein during early development. While mechanistic details of these pathways have primarily been elucidated using the Xenopus model, their roles are conserved in mammals and targeted disruption of key regulators in mouse results in female infertility. Here, we provide a detailed account of the molecular mechanisms involved in translational activation during oocyte and early embryo development, and the role of EPAB in this process.

Subdiffusive Source Sensing by a Regional Detection Method.

Motivated by the fact that the danger may increase if the source of pollution problem remains unknown, in this paper, we study the source sensing problem for subdiffusion processes governed by time fractional diffusion systems based on a limited number of sensor measurements. For this, we first give some preliminary notions such as source, detection and regional spy sensors, etc. Secondly, we investigate the characterizations of regional strategic sensors and regional spy sensors. A regional detection approach on how to solve the source sensing problem of the considered system is then presented by using the Hilbert uniqueness method (HUM). This is to identify the unknown source only in a subregion of the whole domain, which is easier to be implemented and could save a lot of energy resources. Numerical examples are finally included to test our results.

Chaperone-client interactions: Non-specificity engenders multifunctionality.

Here, we provide an overview of the different mechanisms whereby three different chaperones, Spy, Hsp70, and Hsp60, interact with folding proteins, and we discuss how these chaperones may guide the folding process. Available evidence suggests that even a single chaperone can use many mechanisms to aid in protein folding, most likely due to the need for most chaperones to bind clients promiscuously. Chaperone mechanism may be better understood by always considering it in the context of the client's folding pathway and biological function.

Electrostatic interactions are important for chaperone-client interaction in vivo.

It has long been thought that chaperones are primarily attracted to their clients through the hydrophobic effect. However, in in vitro studies on the interaction between the chaperone Spy and its substrate Im7, we recently showed that long-range electrostatic interactions also play a key role. Spy functions in the periplasm of Gram-negative bacteria, which is surrounded by a permeable outer membrane. The ionic conditions in the periplasm therefore closely mimic those in the media, which allowed us to vary the ionic strength of the in vivo folding environment. Using folding biosensors that link protein folding to antibiotic resistance, we were able to monitor Spy chaperone activity in Escherichia coli in vivo as a function of media salt concentration. The chaperone activity of Spy decreased when the ionic strength of the media was increased, strongly suggesting that electrostatic forces play a vital role in the action of Spy in vivo.

Sentinel lymph node biopsy in cutaneous melanoma of the head and neck using the indocyanine green SPY Elite system.

PURPOSE: Lymph node status is the single most important prognostic factor for patients with early-stage cutaneous melanoma. Sentinel lymph node biopsy (SLNB) has become the standard of care for intermediate depth melanomas. Modern SLNB implementation includes technetium-99 lymphoscintigraphy combined with local administration of a vital blue dye. However, sentinel lymph nodes may fail to localize in some cases and false-negative rates range from 0 to 34%. Here we demonstrate the feasibility of a new sentinel lymph node biopsy technique using indocyanine green (ICG) and the SPY Elite near-infrared imaging system. MATERIALS AND METHODS: Cases of primary cutaneous melanoma of the head and neck without locoregional metastasis, underwent SLNB at a single quaternary care institution between May 2016 and June 2017. Intraoperatively, 0.25 mL of ICG was injected intradermal in 4 quadrants around the primary lesion. 10-15 minute circulation time was permitted. SPY Elite identified the sentinel lymph node within the nodal basin marked by lymphoscintigraphy. Target first echelon lymph nodes were confirmed with a gamma probe and ICG fluorescence. RESULTS: 14 patients were included with T1a to T4b cutaneous melanomas. Success rates for sentinel lymph node identification using lymphoscintigraphy and the SPY Elite system were both 86%. Zero false negatives occurred. Median length of follow-up was 323 days. CONCLUSIONS: In this pilot study, Indocyanine green near-infrared fluorescence demonstrates a safe, and facile method of sentinel lymph node biopsy for cutaneous melanoma of the head and neck compared with lymphoscintigraphy and vital blue dyes.

Two pathways of photoproduction of organic hydroperoxides on the donor side of photosystem 2 in subchloroplast membrane fragments.

Earlier the catalase-insensitive formation of organic hydroperoxides (via the interaction of organic radicals produced due to redox activity of P680+· (or TyrZ·) with molecular oxygen) has been found in Mn-depleted PS2 preparations (apo-WOC-PS2) by Khorobrykh et al. (Biochemistry 50:10658-10665, 2011). The present work describes a second pathway of the photoproduction of organic peroxides on the donor side of PS2. It was shown that illumination of CaCl2-treated PS2 membranes (deprived of the PS2 extrinsic proteins without removal of the Mn-containing water-oxidizing complex) (CaCl2-PS2) led to the photoproduction of highly lipophilic organic hydroperoxides (LP-OOH) (in amount corresponding to 1.5 LP-OOH per one reaction center of PS2) which significantly increased upon the addition of exogenous electron acceptor potassium ferricyanide (to 4.2 LP-OOH per one reaction center). Addition of catalase (200 U/ml) before illumination inhibited ferricyanide-induced photoproduction of hydroperoxides while no effect was obtained by adding catalase after illumination or by adding inactivated catalase before illumination. The hydroperoxide photoproduction was inhibited by the addition of exogenous electron donor for PS2, diphenylcarbazide or diuron (inhibitor of the electron transfer in PS2). The addition of exogenous hydrogen peroxide to the CaCl2-PS2 led to the production of highly lipophilic organic hydroperoxides in the dark (3.2 LP-OOH per one reaction center). We suggest that the photoproduction of highly lipophilic organic hydroperoxides in CaCl2-PS2 preparations occurs via redox activity of H2O2 produced on the donor side of PS2.

Spy1 Protein Mediates Phosphorylation and Degradation of SCG10 Protein in Axonal Degeneration.

Axon loss is a destructive consequence of a wide range of neurological diseases without a clearly defined mechanism. Recent data demonstrate that SCG10 is a novel axonal maintenance factor and that rapid SCG10 loss after injury requires JNK activity; how JNK induces degradation of SCG10 is not well known. Here we showed that SCG10 was a binding partner of Spy1, a Speedy/RINGO family protein, which participated in cellular response to sciatic nerve injury. During the early stage of axonal injury, Spy1 expression was inversely correlated with SCG10. Spy1 mediated SCG10 phosphorylation and degradation partly in a JNK-dependent manner. Inhibition of Spy1 attenuated SCG10 phosphorylation and delayed injury-induced axonal degeneration. Taken together, these data suggest that Spy1 is an important regulator of SCG10 and can be targeted in future axo-protective therapeutics.

Equivalence of MammaPrint array types in clinical trials and diagnostics.

MammaPrint is an FDA-cleared microarray-based test that uses expression levels of the 70 MammaPrint genes to assess distant recurrence risk in early-stage breast cancer. The prospective RASTER study proved that MammaPrint Low Risk patients can safely forgo chemotherapy, which is further subject of the prospective randomized MINDACT trial. While MammaPrint diagnostic results are obtained from mini-arrays, clinical trials may be performed on whole-genome arrays. Here we demonstrate the equivalence and reproducibility of the MammaPrint test. MammaPrint indices were collected for breast cancer samples: (i) on both customized certified array types (n = 1,897 sample pairs), (ii) with matched fresh and FFPE tissues (n = 552 sample pairs), iii) for control samples replicated over a period of 10 years (n = 11,333), and iv) repeated measurements (n = 280). The array type indicated a near perfect Pearson correlation of 0.99 (95 % CI: 0.989-0.991). Paired fresh and FFPE samples showed an excellent Pearson correlation of 0.93 (95 % CI 0.92-0.94), in spite of the variability introduced by intratumoral tissue heterogeneity. Control samples showed high consistency over 10 year's time (overall reproducibility of 97.4 %). Precision and repeatability are overall 98.2 and 98.3 %, respectively. Results confirm that the combination of the near perfect correlation between array types, excellent equivalence between tissue types, and a very high stability, precision, and repeatability demonstrate that results from clinical trials (such as MINDACT and I-SPY 2) are equivalent to current MammaPrint FFPE and fresh diagnostics, and can be used interchangeably.

Folding mechanisms of periplasmic proteins.

More than one fifth of the proteins encoded by the genome of Escherichia coli are destined to the bacterial cell envelope. Over the past 20years, the mechanisms by which envelope proteins reach their three-dimensional structure have been intensively studied, leading to the discovery of an intricate network of periplasmic folding helpers whose members have distinct but complementary roles. For instance, the correct assembly of ß-barrel proteins containing disulfide bonds depends both on chaperones like SurA and Skp for transport across the periplasm and on protein folding catalysts like DsbA and DsbC for disulfide bond formation. In this review, we provide an overview of the current knowledge about the complex network of protein folding helpers present in the periplasm of E. coli and highlight the questions that remain unsolved. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.