The Internet of Things (IoT) is an emerging paradigm that proposes the connection of objects to exchange information in order to reach a common objective. In IoT networks, it is expected that the nodes will exchange data between each other and with external Internet services. However, due to deployment costs, not all the network devices are able to communicate with the Internet directly. Thus, other network nodes should use Internet-connected nodes as a gateway to forward messages to Internet services. Considering the fact that main routing protocols for low-power networks are not able to reach suitable performance in the displayed IoT environment, this work presents an enhancement to the Lightweight On-demand Ad hoc Distance-vector Routing Protocol—Next Generation (LOADng) for IoT scenarios. The proposal, named LOADng-IoT, is based on three improvements that will allow the nodes to find Internet-connected nodes autonomously and dynamically, decreasing the control message overhead required for the route construction, and reducing the loss of data messages directed to the Internet. Based on the performed assessment study, which considered several number of nodes in dense, sparse, and mobility scenarios, the proposed approach is able to present significant results in metrics related to quality-of-service, reliability, and energy efficiency. The Internet of Things (IoT) is a wide concept that has attracted attention from the research community in recent years . The terIoT devices form, in general, a low power and lossy network (LLN), composed of many nodes with strong restrictions on memory, processing capacity, and, in some cases, energy. Depending on the application, the nodes in an IoT network can have different hardware capacities and application objectives. In the IoT scenario presented in In the context of low-power IoT networks, the routing protocols can be grouped into two types based on the route creation principles: proactive and reactive . ProactiLOADng-IoT improves network QoS and reliability by increasing the packet delivery ratio and the reduction of end-to-end latency for the different message types exchanged by nodes in both dense and sparse IoT scenarios.It reduces the number of control messages required to construct routes among nodes, contributing to a more efficient network with lower overhead.LOADng-IoT reduces the amount of energy required to both build paths and route data messages, making the network more power efficient.It dispenses with the use of predefined Internet gateways since the Internet-connected nodes are sought on demand and can change according to their connection availability. This feature also removes the existence of a single point of failure (SPOF) for the connection of IoT devices with external Internet services.It presents a flexible solution, whereby parts of the proposal can be adopted according to the hardware capacities of the nodes.Although it uses the most indicated route creation principles in the IoT scenario considered in this study, LOADng can present several problems such as the necessity of previous and static definitions of the nodes responsible for providing the Internet connection to other network devices. Also, the obligation of several routing creation processes to construct on-demand routes to P2P traffic can provoke a high control message overhead. Thus, the main objective of this work is to create an enhancement for LOADng to allow the protocol to better discover and maintain routes for traffic directed to the Internet in IoT networks formed by devices with different capacities. The proposed approach, LOADng-IoT, is composed of three improvements that are able to boost the process of route discovery, reduce the overhead of control messages, and improve the network’s quality-of-service (QoS). In summary, the proposal allows the nodes to find Internet-connected nodes without the prior definition of a gateway. This behavior allows nodes without an Internet connection to forward their data packets to external Internet services with much greater reliability and lower latency. Also, the proposed solution presents a cache system for routes to Internet nodes to reduce the control message overhead required in the process of route discovery. Finally, the solution introduces a new error code that should be used to avoid the loss of data packets to the Internet. Thus, the main contributions of the proposal presented in this work (LOADng-IoT) are as follows:The remainder of this document is organized as follows. This work proposes a new enhancement to aid the LOADng route discovery process in IoT scenarios composed of nodes with different capacities and variable message traffic. In the current literature, several studies focus on performance and propose improvements for LOADng. However, to the best of the authors’ knowledge, the current related literature does not propose improvements to the route discovery process of Internet nodes for LOADng in IoT scenarios.In , the autBased on the limited performance of LOADng in MP2P scenarios, Yi and Clausen  proposedA new composite routing metric for LOADng is proposed in . The terA multipath improvement for LOADng is also proposed in . The NeiAraújo et al.  propose weakLinks mechanism to identify links with low quality across a path. In the route creation process, paths with a high number of weakLinks are avoided. The choice of the best route also considers the residual energy of the nodes and hop count. In all the studied scenarios, REL was able to outperform the AODV regarding latency, packet delivery ratio, and network lifetime.A routing protocol for IoT networks based on the composition of routing metrics is presented in . The RouAraújo et al.  present In ,23, the Considering the limitations of the current literature and the requirements of IoT low-power networks, this work proposes a new mechanism for LOADng that will allow it to search Internet-connected nodes in a dynamic and on-demand manner. Moreover, the proposed approach can improve normal data traffic among the nodes, enabling the network to become more energy efficient and reliable.The LOADng routing protocol proposes a simplification of the AODV , a well-The following subsections present an overview of the LOADng protocol. These explanations are necessary to understand the approach proposed in this work.LOADng is a reactive routing protocol based on route discovery using route request and route reply messages. Thus, when a node wants to send a data message and the route to the destination is unknown, it should begin a new route discovery process. To this end, the node broadcasts a route request (RREQ) message to search for a route to the desired destination. Each node that receives an RREQ should perform message processing and consider the message to be forwarded. This process continues until the RREQ reaches the sought destination. The destination should then generate a route reply (RREP) message to answer the received RREQ. The RREP is forwarded in unicast to the RREQ originator, constructing a route between the two nodes interested in the message exchange. Finally, the RREP is received by the RREQ originator, which should begin to send data messages using the path created by the route discovery process.The process of route discovery is performed with the use of control messages inspired by AODV. RREQ messages are always used to request the creation of a route to a destination when a node needs to send a data message and the path to the destination is unknown. RREP messages are used by the destination that receives the RREQ as an answer to the request for route creation. An RREP message may, optionally, require an acknowledgment. In this case, the route reply acknowledgment (RREP_ACK) message is used to answer a received RREP. When a node fails at the moment of data message forwarding, a route error (RERR) message can be used to inform the data message originator of the problem detected. The RERR can also be used when the data message destination is unknown by the intermediate node. ackrequired defined as true. In the process of route discovery, control messages are used in conjunction with an Information Base maintained by each network node. According to the content of the control messages, the Information Set of nodes are fed and updated. The main elements of the Information Base are the following: Routing Set, Blacklisted Neighbor Set, and Pending Acknowledgment Set. The Routing Set is composed by route tuples entries that store data about the neighbor nodes. Based on the Routing Set, a node can verify the existence of a path to a destination or the necessity of starting a new route discovery process. The Blacklisted Neighbor Set is responsible for storing the addresses of nodes with possible communication inconsistencies that make the bidirectional linkage unavailable. The Pending Acknowledgment Set records information about the RREP messages sent with the field When a node wants to send a data message, it should look for a route to the message destination on its Routing Set. If the path is found, the node should forward the message to its destination through the next hop node. The message forwarding process is described in detail in originator and the address of the desired destination in the destination field. It should also set a unique seq-num to the RREQ and define the other message fields. Then, the node should broadcast the generated RREQ to its neighbors.In the routing discovery process, the node generates a new RREQ message, defining itself as hop-count, hop-limit, and route-metric from the message. In sequence, the node should search for a route entry for the message originator on its Routing Set. If the route is not found, a new route entry for the message originator is created. Then, the created or found route entry is compared with the fields of the received message to verify whether or not the message can be used to update the route entry. If the message is valid, the route entry is updated, the common processing is finished, and the message returns to its specific processing. In contrast, if the message is not used to update the route entry, the node should verify the message type, send an RREP_ACK if required, and drop the message. When the message returns to the specific RREQ processing, the node should check whether it is a message destination. If negative, the node should verify whether the message is valid to be forwarded (checking the hop-count and hop-limit), updating its fields, and forward it using broadcast. Otherwise, if the node is the RREQ destination, it should generate an RREP message to answer the request from the RREQ originator.Each receiver of the RREQ message should execute its processing according to the flowchart presented in destination, the address of its originator in the originator field, and a unique seq-num. After being generated, the RREP should be sent in unicast to its destination. Thus, the RREP originator should look for a route entry to the destination on its Route Set and forward the message to the R_next_hop node. Note that the route entry should be found after it has been created by the RREQ message being received. A node that receives the RREP message should perform its processing as described in the flowchart in R_next_hop node using unicast. Otherwise, if the node is the RREP destination, the route discovery process is completed, and the data message can be sent using the constructed path.The generated RREP message should have the address of the RREQ originator as In the data message sending process, the node should use the path created in the route discovery process to deliver the data message to its correct destination. Thus, the node consults its Routing Set looking for an entry that matches the message destination. In sequence, the node should forward the message to the next hop of the found route entry. According to the latest LOADng specification, a node should always refresh the valid time of a route entry that it uses. The intermediate node that receives a data message should forward to the next hop of the path based on the information in its Routing Set. This process occurs until the message reaches its final destination. If an intermediate node does not find a route entry that matches the message destination, it should perform a new route discovery process to recover the broken path. If the path recovery does not succeed, the node should generate an RERR message to inform the data message originator of the impossibility of delivering the message successfully.smart-rreq flag set as true. Every node that receives a SmartRREQ (RREQ message with smart-rreq true) should perform additional processing in the RREQ message handling. After executing all the initial processing, and after verifying whether the message is valid for forwarding, the node should perform the specific processing of SmartRREQ. Thus, the node checks whether it owns a route entry on its Routing Set to the message destination with R_next_hop that is different from the previous hop of the received SmartRREQ. If this condition is satisfied, the node should transmit the SmartRREQ message in unicast to the next address found. The next hop that receives the SmartRREQ message should perform the same processing until the message reaches the final destination. If a node does not find a route entry to the SmartRREQ destination, the message should be forwarded using broadcast. The destination of a SmartRREQ should answer the request by generating a normal RREP. Hence, the SmartRREQ enhancement can reduce the number of broadcast transmissions, thereby contributing to reducing the control message overhead required to discover a new route and decreasing the network energy consumption.To reduce the number of control messages exchanged during the route discovery process, the SmartRREQ enhancement was proposed for LOADng . With thThis work proposes an enhancement for the LOADng protocol in IoT networks composed of devices with different capabilities. The proposed LOADng-IoT introduces a new route discovery process dedicated to finding devices with the capacity to forward special messages from other nodes. The following subsection explains the IoT applications scenario in which the proposed approach can be applied. In the sequence, LOADng-IoT is fully described, including its features, requirements, and operation.This work considers an IoT network as presented in A smart home (SH) IoT application can be used to exemplify the use of the presented network model. In a SH, the smart objects with a low necessity of consulting external Internet services  can be represented by simple nodes. In contrast, smart objects that require continuous Internet-connection  can be represented by INs linked to the Internet using cellular network or optical fiber. Thus, as an example, a smart window can occasionally consult an external Internet service to verify the weather forecast. The smart window, to access the service, should use an IN as its gateway to the Internet. Besides, in a context of a local message exchange, an air-conditioner, when activated, can send a local message to close the smart windows without the necessity of using the Internet to perform this communication.Current routing solutions can address the presented network model when the simple nodes have been previously configured with a default gateway to forward their Internet messages. Thus, prior knowledge of the nodes with Internet connection capacity is required to then define a gateway for each simple node. This approach, although functional, can give rise to several issues. The obstacles that can occur using this simplistic approach are identified as: (i) high deployment time is required to define the gateway of each simple node; (ii) Internet-connected nodes can be overloaded with Internet messages from simple nodes; (iii) bad deployment can make simple nodes create long paths to their gateways; (iv) simple nodes can become unable to send Internet messages if their gateways lose Internet connectivity.Based on the exposed constraints, and seeking to better address the requirements of the described IoT network scenario, this work proposes a new enhancement for the LOADng protocol that is able to optimize the route discovery process for INs and improve the network performance. The proposed LOADng-IoT can simplify the discovery of Internet routes, avoiding the necessity of the prior definition of gateways for Internet messages. In addition, the proposal reduces the number of control messages required to construct paths to INs and makes the data message forwarding process more reliable. The following subsections present a detailed description of the proposed LOADng-IoT.The proposed LOADng-IoT is composed of three components: the Internet route discovery process, the Internet Route Cache (IRC) system, and a new error code for RERR messages. The first component is responsible for finding IN nodes without the requirement of previous knowledge of its address in the local network. Thus, a node that wants to send a message to the Internet should start an Internet route discovery process by broadcasting a special RREQ, named RREQ-IoT. The message has the objective of seeking an IN that can be used as a gateway for the RREQ-IoT originator. To reduce the number of broadcast transmissions, an intermediate node that knows a route for an IN can forward the RREQ-IoT message to it using unicast transmission (in the same way as SmartRREQ). When an IN receives an RREQ-IoT message, it should generate a special RREP to answer the request. This message, named the RREP-IoT, is forwarded in unicast via the opposite route created by the RREQ-IoT. Each node that receives an RREP-IoT should create an entry on its Routing Set with the information that the message originator has an Internet connection. When the RREP-IoT reaches its destination, the node should immediately start to send the Internet data messages. The proposed Internet route discovery process of LOADng-IoT is fully described in The second component is responsible for storing the Internet routes (routes to Internet-connected nodes) removed from the Routing Set. During the Internet route discovery process, the nodes create entries on the Routing Set to the INs. These entries, which have a valid time, can expire and be removed from the Routing Set when not used. Thus, to reduce the number of transmissions in a new Internet route discovery and to allow the nodes to follow a previously known Internet route, these entries, when removed, have some of its information inserted in a new data structure, the IRC. The IRC should always be consulted when a new Internet route discovery process is started and should, when possible, indicate a previously known Internet route to direct the discovery process. The IRC is optional and should be adopted according to the hardware capacity of the network devices. A complete description of the proposed IRC is presented in INTERNET_CONN_LOST error code should be used to indicate to an Internet data message originator that it was not possible to forward the message to the Internet. Thus, the receiver of the RERR should start a new Internet route discovery process to find a new IN to transmit its Internet messages. The generation, processing, and functioning of the proposed new error code for RERR messages are detailed in The third component is a new error code for RERR messages. The The proposed LOADng-IoT requires several increments in the existing default LOADng structure. In addition to the increments previously presented, the LOADng-IoT also presents several new features required to improve the performance of the studied IoT networks. iot set as TRUE . Then, the node defines its own address as the RREQ-IoT originator and destination, and a set new unique seq-num. In the following, the node should consult its IRC (if in use) to verify the existence of a previously known Internet route. At this point, the IRC is considered to be empty, and will be explored in the next subsection. Thus, the node should transmit the generated RREQ-IoT in a broadcast to its neighbors.The Internet route discovery should always be initiated when a node does not find a route entry to an IN on its Routing Set. Thus, the node should create an RREQ with the flag R_Internet_conn set as FALSE. In addition, if the received RREQ-IoT is used to update the information on an existing route entry, the R_Internet_conn should never be changed to false. If the common processing is concluded without the message being dropped, the message handling proceeds to the next step. Thus, in the specific RREQ-IoT processing, the node should check whether it has an active Internet connection . If the node identifies an active Internet connection, it should reply to the RREQ-IoT by generating and transmitting an RREP-IoT message to the request originator. However, if the node does not have an Internet connection, it should check whether the message is valid for forwarding. If true, the node searches for an Internet route on its Routing Set. Thus, if it is found, the node should use a mechanism similar to SmartRREQ to assist the path creation. Hence, the node changes the RREQ-IoT destination to the found R_destination, updates the other message field and sends the message in unicast to the R_next_hop. If more than one Internet route exists in the Routing Set, the node should select the one that best address the used routing metric. However, if an Internet route entry is not found in the Routing Set, the node should use the IRC mechanism, if it is activated. Considering that the node does not use the IRC, it should update the RREQ-IoT message fields and transmit the message in broadcast. Independent of the transmission mode of the RREQ-IoT (unicast or broadcast), the next receiver of the message should perform the process previously described.Each node that receives an RREQ-IoT should perform its processing according to the flowchart in A needs to send an Internet message and has begun an Internet route discovery process. Node B has received the RREQ-IoT from A and begun its processing. B does not have an Internet connection but finds an Internet route to node C on its Routing Set. Thus, B changes the RREQ-IoT destination to node C and forwards the message in unicast to C. Thus, inspired by the SmartRREQ, the Internet route discovery process is optimized to reduce the number of broadcast transmissions, thereby contributing to the reduction of energy consumption.Please note that unlike the normal route discovery process, the Internet route discovery process searches for any IN rather than a defined destination address. During the RREQ-IoT message processing, the node does not verify whether it is the message destination, but whether it has an active Internet connection. In addition, it is possible that an intermediate node that knows an Internet route will change the RREQ-IoT destination and redirect the Internet route discovery. originator, and the address of RREQ-IoT originator as destination. The message should also have a new unique seq-num. Then, the node transmits the RREP-IoT message in unicast to the next address in the path to the destination.As previously explained, a node with an Internet connection that receives an RREQ-IoT should generate a reply message to answer the request. Thus, the node generates an RREP-IoT, defines its own address as R_Internet_conn is TRUE. In addition, the R_valid_time of the Internet route should be, by default, two times greater than the valid time of regular routes. Since only Internet-connected nodes can generate RREP-IoT messages, the intermediate nodes that forward RREP-IoT messages should not be included as R_Internet_conn set as TRUE. Thus, the RREP-IoT message performs the maintenance of routes and creates the path to the Internet nodes. At the end of the common processing, the node can generate an RREP_ACK message, if necessary. In the LOADng-IoT, the RREP_ACK generation, transmission, and processing are equal to the default LOADng. In sequence, the RREP-IoT receiver verifies whether it is the message destination. If false, the node should check whether the message is valid to forward, perform the message update, and transmit it to its destination. Otherwise, if the node is the message destination, the Internet route discovery process is completed, and the node can begin the sending of Internet messages.The node that receives an RREP-IoT should perform its processing, which is very similar to the normal RREP processing, following the flowchart in In the described Internet route discovery process, all nodes that receive an RREQ-IoT should reply using an RREP-IoT. Thus, it is possible for more than one RREP-IoT to be received by the request originator. This behavior makes the construction of several Internet routes possible, one for each different IN. Hence, a node that intends to send an Internet message should look up its Routing Set and find the best path among those available. The selection of the best Internet route should be made based on the used route metric or the lowest number of hops. The process of Internet message sending and forwarding is described in detail in In the course of the network functioning, all route entries of the Routing Set can be removed when the valid time expires. This process occurs to allow the nodes to reduce the memory usage and make the creation of paths to other nodes possible. In the LOADng-IoT, the valid time of the regular routes and the Internet routes can be different and should be adjusted according to the expected traffic in the network. However, considering a scenario where both message types are equality generated, an Internet route tends to be used more by representing a path to all Internet messages. Thus, by default, the authors suggest that the Internet routes have a valid time that is two times greater than regular routes.Even with a higher valid time, Internet routes that are not used can expire and be removed from the Routing Set of the nodes. Thus, when a node needs to send a new Internet message, the whole Internet route discovery process should be completed again, transmitting several control messages and expending more network resources. To reduce this problem, LOADng-IoT offers an optional improvement that is able to minimize the control message overhead during the construction of Internet routes. This mechanism, the IRC, stores the most relevant information about the last Internet route entries removed from the Routing Set. Then, when it is necessary to perform an Internet route discovery, the node should check its IRC to verify the existence of a previously known Internet route. If positive, the node can direct the Internet route discovery to the destination of the found entry in the IRC.R_Internet_conn is TRUE), its R_next_addr and R_dest_addr are used to create a new route cache entry that is inserted in the IRC set. As presented in An entry can be removed from the Routing Set due to valid time expiration or to lack of memory for the insertion of a new entry. Thus, with the use of the IRC, if the removed entry is an Internet route . B receives the RREQ-IoT from A and realizes its processing normally. However, B, at the moment of checking its Routing Set, finds an Internet route to node C. Thus, B changes the received RREQ-IoT destination to C and forwards the message in unicast. Node C then receives the request from A and sends a reply offering the required Internet route. This behavior is accepted because the intention of an RREQ-IoT is to reach a route to the Internet, independent of the IN providing the connection. In addition, this redirection of the RREQ-IoT ensures that the Internet route discovery process follows with most recent information (considering that the information provided by the Routing Set is frequently newer than the information provided by the IRC). However, if an Internet route is not found in the Routing Set of the intermediate node, the intermediate node should verify its IRC, change the message destination (if necessary), and then forward the message in unicast to an IN that is able to provide an Internet connection to the RREQ-IoT originator. Finally, whether the IRC set is empty, the node should change the destination address of the RREQ-IoT to the same address as its originator and send the message in broadcast. This process “converts” the RREQ-IoT received in unicast in a normal RREQ-IoT to be broadcasted and the Internet route discovery process can continue until an IN is reached.This situation is exemplified in The use of the IRC mechanism allows the nodes to reduce the number of control messages required to construct an Internet route. The cache mechanism is used to direct the RREQ-IoTs to a previously known IN. Thus, when adopted, the IRC contributes to the reduction of the number of packet collisions, minimizes energy consumption, and improves network efficiency. As explained, the entries in the IRC set are only removed by lack of memory or the reception of an RERR message. This process is discussed in the description of the new error code proposed in this work in INTERNET_CONN_LOST), is defined by code 253. The used code number is included in the range of experimental use codes according to the most recent LOADng [Due to unexpected situations, the Internet nodes can sometimes lose Internet connection. To avoid these nodes receiving Internet messages when they have no connection, this paper proposes a new error code with the function of advising the neighbor nodes that the Internet connection has been lost. The error code, described as “Internet connection lost” . However, to send an Internet message, the node should to select the best path based on the selected routing metric since the Internet route discovery process can create several routes to different INs. Thus, it is possible to choose the best path among those available. The nodes that receive a data message (both simple and Internet) should consult its Routing Set to find the next hop to the destination node. This process should continue until the data message is delivered.Independent of the message type, it is possible for a broken route to occur during the message forwarding process. In this case, the intermediate node that was not able to forward the message detects the broken path, queues the data message, and starts a new route discovery process according to the message type. If the message is simple, the node should begin a normal route discovery using the standard LOADng procedure. However, if the message is directed to the Internet, the node should start an Internet route discovery following the process described in This section presents the performance assessment realized to evaluate the behavior of the proposed solution. The Cooja simulator/emulator, which is a part of the Contiki O.S. , was usenxn nodes. The simulated area grew together with the number of nodes. Thus, a fixed network density was maintained where the nodes had between two and four neighbors.Grid Sparse Scenarios: the network nodes were organized in linear grids of Random Dense Scenarios: the different quantity of nodes was randomly deployed in an area of 200 square meters just once. Thus, the random deployments were the same for all compared proposals. The simulated area was the same for the different quantities of nodes. Hence, the network density grew with the increase in the number of nodes.Mobility Dense Scenarios: the nodes were deployed with the same positions of random dense scenarios in an area of 200 square meters. However, the nodes with an Internet connection was able to move in the whole area of the studied environment.The proposed LOADng-IoT was compared with the most recent version of LOADng and LOADng-SmartRREQ. The objective was to analyze the behavior of the proposed solution with the other proposals in different scenarios. Thus, situations were created with three topology organizations: grid sparse, random dense, and mobility dense. For all the topologies, the number of nodes in the network changed from 16 to 64. This quantity was chosen because it can represent the majority of the existing small-scale IoT application scenarios, mainly in smart homes. The following itemization presents more details on the used grid, random, and mobility topologies.For all scenarios and the different quantities of nodes, the simulation time was 600 s. In the application, all network nodes generated and sent data messages in variable intervals of between 10 and 15 s. The minimum data message interval was defined as 10 to avoid the nodes being overloaded with several data messages while they were still realizing a route discovery process. This measure was required because the nodes do not implement a significant buffer for data messages in the routing layer. Thus, all data messages generated inside a route discovery process are lost. The data message generation should be able to address the requirement of Equation , the energy spent per delivered data bit (AES), the control message overhead per delivered data message (CMO), and the percentage of packets with low latency (PLL). For all studied metrics, the simulations were executed 30 times, and the results presented a confidence interval of 95%.N is the number of nodes in the network, i, and i.The PDR metric represents the number of data messages that were successfully delivered to the destination node. Thus, a high PDR represents an efficient network that is able to deliver the generated data messages with high reliability. This metric is constantly affected by the quality of the links among the nodes, the radio interference provoked by neighbor devices, and collisions with other data and control messages. In this paper, the Internet messages delivered for a node without an Internet connection were considered lost. The PDR value of the network was obtained according to Equation :(2)PDR=∑N is the number of nodes in the network, i, i.The average energy spent per delivered data bit metric represents the amount of energy spent by the network to successfully delivery each data bit to its destination. Thus, the less energy spent to deliver the data successfully, the higher the power efficiency of the network. The results obtained by this metric are affected by the energy consumption of the nodes and the packet delivery ratio. The metric is computed using Equation :(3)AES=∑N is the number of nodes in the network, i, and i.The control message overhead per delivered data message (CMO) metric shows the number of control message transmissions required to deliver each data message successfully. As in the previously presented metric, the results of the CMO are directly related to the packet delivery efficiency. Although control messages are not used during the forwarding of data packets, it is possible to count the overhead generated by the nodes to construct the path used to deliver the data messages. Thus, calculating the ratio between the quantity of control message transmission used to discover the routes and the number of data packets delivered, it is possible to obtain the mean of control message transmissions required to deliver each data message. Thus, this metric is calculated according to Equation :(4)CMO=∑The results obtained for the CMO metric are presented in N is the number of nodes in the network, i, The metric of the percentage of packets with low latency exposes the percentage of data packets delivered with a latency that is considered low and acceptable for low-power devices in IoT applications. Several aspects can affect the metric results; among these, it is possible to cite: the length of the path constructed during the route discovery process; the quality of links among the nodes; the radio duty cycle frequency; the medium access control protocol; the density of nodes, etc. The PLL metric is computed according to Equation :(5)PLL=∑This work presents a new improvement to the LOADng routing protocol in IoT scenarios when the network devices have different capacities and use different message types. The proposal was compared with the default implementation of LOADng and LOADng-SmartRREQ through simulations using COOJA. Four different metrics were studied to expose the network performance in terms of reliability, QoS, and power efficiency. For all the considered metrics, LOADng-IoT demonstrated better performance for sparse, dense, and mobile networks. These significant results were obtained due to the set of improvements provided by the proposed approach.Unlike other approaches, the proposed LOADng-IoT does not require a previous definition of gateways and, hence, it can find the most appropriated Internet node to forward messages. This feature provides a self-adaption capacity to the network nodes and reduces the necessity of human intervention in both network deployment and execution. Optionally, LOADng-IoT allows the use of a cache system dedicated to storing Internet routes, enabling the nodes to direct the route discovery to INs that anteriorly had been used as gateways. These approaches together permits LOADng-IoT to reduce the control message overhead required to find the Internet routes, contributing to the reduction of power consumption and improving the packet delivery ratio. Finally, this work proposed a new error code that makes it possible for the Internet nodes to advise the other network nodes about its temporary Internet connection loss. Thus, devices that intend to send Internet messages can find new gateways to forward messages, increasing the chances of successful delivery. It was also noted that LOADng-IoT performance was, in general, less variable when compared with the other studied proposals. This behavior is justified because LOADng-IoT requires fewer radio transmissions to perform the data packet delivery and route discovery. Thus, LOADng-IoT is less affected by the interferences and collision that commonly makes the network performance unstable. In conclusion, the authors deduce that, together, all of the proposed mechanisms that comprise LOADng-IoT provide a new significant enhancement for IoT networks, allowing them to attain better QoS, efficiency, and reliability.For future work, the authors suggest that experiments in real IoT environments should be conducted to test the results obtained using computational simulation. Moreover, the source code from the proposed solution should be improved, documented, and disseminated to the scientific community.


In sub Saharan Africa one of the key challenges in assessment using neuropsychological tools has been the lack of adequately validated and easily implementable measures. This study will translate into English, adapt and standardize the Computerized Battery for Neuropsychological Evaluation of Children (BENCI). The BENCI battery will be adapted using back-translation design, comprehensive cultural adaptation and standardized in a case–control study involving two groups of children: HIV infected and HIV unexposed, uninfected children. The content adaptation will be iteratively carried out using knowledge of English and feedback from pilot testing with children. The proposed study will first involve the cultural adaptation of the BENCI. It will then recruit 544 children aged 8–11 years with half of them being HIV+, while the other half will be HIV unexposed-uninfected. Test–retest reliability will be analyzed using Pearson’s correlation while ANOVA and correlational analyses will be used to calculate discriminant, convergent and construct validity.This study will result in an open access adequately adapted and standardized measure of neuropsychological functioning for use with children in East Africa. The protocol paper provides an opportunity to share the planned methods and approaches. Children growing up in low and middle-income countries (LAMICs) are at a significant risk of experiencing neurocognitive impairment due to exposure to multiple risk factors . HoweverRecent efforts indicate that using systematic adaptation process can contribute to the development of neuropsychological measures that can be adequately used in LAMICs settings . We woulOne attractive feature of the BENCI is its computerized nature which makes it relatively easy to administer and get results readily and paper-free. The BENCI has good psychometric properties in terms of validity and reliability . It has To evaluate the internal consistency, and re-test reliability of the BENCI;To evaluate the construct and criterion validity of these measures;To evaluate discriminative validity of the measures by comparing the performance of HIV infected and HIV exposed uninfected school going children.The general objective of this study is to establish the reliability and validity of the BENCI and its utility in monitoring outcomes among HIV positive school going children. The specific objectives are:A three-phased approach will be carried out. In the first phase, the linguistic and semantic equivalence of the BENCI content will be ensured through back translation design from Spanish to English by two translators. An evaluation of the tools structure and appropriateness will also be done where psychologists will check for the appropriateness of the pictures and other materials. In the second phase, a pilot study among 10 children will evaluate the appropriateness of the items including pictures and instructions. While in the third phase, the psychometric properties of the English version of the BENCI with regards to the Kenyan population of HIV+ children will be evaluated in a case control study at a HIV programme and three public schools.One of the study sites will be a county level HIV programme, which is an outpatient programme catering for HIV infected individuals and their families from culturally diverse backgrounds within resource poor settings.The normative data will be collected from 3 primary schools in the resource poor setting in Nairobi County.The two settings are in the Kenyan capital city of Nairobi which has a high level of literacy 87.1%) with English being the primary language of instruction in the schools [7.1% withA socio-demographic questionnaire will incorporate elements such as age, gender and education background among other variables.A breakdown of measures within each cognitive domain within BENCI is available in Table The Kilifi Neuropsychological Tool Kit comprises of a set of measures adapted and standardized from published measures. The tools have good psychometric properties with split half reliability being between .70 and .84 while internal consistency was ≥ .70 . Some ofThe Guidelines for Translating and Adapting Tests developed by the International Test Commission will be used in adapting and standardizing the tool . PermissA pretest of the BENCI will be carried out in order to identify elements that may not be well understood by respondents and problems that may be encountered during the main study. The piloting will be carried out among randomly selected 10 children from a community-based HIV programme. The randomization will be carried out among 8- to 10-year-old who are living with HIV. They will be randomly selected as they come into the clinic for their usual appointments and requested to enroll for the pilot study. The piloting will aid in adapting BENCI in terms of modifying item formats that may not be recognized by respondents, eliminating translation bias among other modifications. In order to improve content validity, inter-rater reliability analysis will be carried out where two raters will review the results of the pretest. One rater will administer the tool amongst the pilot sample, while the other rater will review how the tool is administered and the respondent responds. This work will be qualitative in nature trying to identify and refine the items and their relevance within BENCI.HIV exposed and unexposed children will be considered as comparative groups—and potentially matched on the patient background characteristics (age and gender). The two comparable groups will be of equal sample sizes which will be calculated using a formula cited in Wittes .2 + 63.72)/2) = 66.3. Together with a significance level of 5% and a power of 80%, these result in a total sample size of 544 respondents, 272 in each study arm. So, the study will need to enroll 272 HIV exposed children and 272 unexposed children.In this study sample size computation is based on data from earlier studies in Africa, the nexposed . Thus, tRespondents will be recruited using random stratified sampling by sex and age. A list of all respondents aged 8–11 years in the HIV program and 8–11 years in the primary schools according to gender will then be extracted from the children data base in the institutions. The children’s caregivers will then be requested to give consent on behalf of the children in the schools and the HIV program. A list of all the children with parental consent will then be compiled in readiness for data collection day. On data collection day, the respondents who agree to participate, will be shown the room with neuropsychological assessment tools and proceed with the demographic questionnaire and later on the neuropsychological tests. The process will take around 90–120 min per child.Since the data generated by the BENCI will be collected in computerized format, it will automatically be numerically coded with identifiers attached to different respondents and excel sheets will be generated as programmed in the tool. The Kilifi Toolkit subsets data will be manually keyed into Excel sheets after being numerically coded with identifiers that are attached to differentiate the respondents. Descriptive statistics, as well as, frequency distributions will be used to analyze the demographic traits among other characteristics of the HIV uninfected unexposed (control) and HIV infected  with the aid of SPSS. Intraclass correlation, in the same software, will be used to calculate test–retest reliability. To examine convergent validity, the raw scores of BENCI subtests will be compared to the raw scores of the subtests in Kilifi Toolkit. A confirmatory factor analysis will be used to assess construct validity. The validity indicators will be several alternative fit statistics as recommended by Hu and Bentler : Chi squThe study will be conducted in a community setting; hence the findings may not be replicated within a clinical setting.


Epidemiological evidence suggests that cadmium (Cd) is one of the causative factors of prostate cancer, but the effect of Cd on benign prostatic hyperplasia (BPH) remains unclear. This study aimed to determine whether Cd exposure could malignantly transform BPH1 cells and, if so, to dissect the mechanism of action. We deciphered the molecular signaling responsible for BPH1 transformation via RNA-sequencing and determined that Cd induced the expression of zinc finger of the cerebellum 2 (ZIC2) in BPH1 cells. We noted Cd exposure increased ZIC2 expression in the Cd-transformed BPH1 cells that in turn promoted anchorage-independent spheroids and increased expression of stem cell drivers, indicating their role in stem cell renewal. Subsequent silencing of ZIC2 expression in transformed cells inhibited spheroid formation, stem cell marker expression, and tumor growth in nude mice. At the molecular level, ZIC2 interacts with the glioma-associated oncogene family (GLI) zinc finger 1 (GLI1), which activates prosurvival factors , as well as an X-linked inhibitor of apoptosis protein (XIAP)) signaling in Cd-exposed BPH1 cells. Conversely, overexpression of ZIC2 in BPH1 cells caused spheroid formation confirming the oncogenic function of ZIC2. ZIC2 activation and GLI1 signaling induction by Cd exposure in primary BPH cells confirmed the clinical significance of this oncogenic function. Finally, human BPH specimens had increased ZIC2 versus adjacent healthy tissues. Thus, we report direct evidence that Cd exposure induces malignant transformation of BPH via activation of ZIC2 and GLI1 signaling. Both BPH and prostate cancer (CaP) share similar etiology and pathophysiological factors5. Although, BPH to CaP conversion is controversial, the published evidence suggests that BPH patients have a two to threefold increased risk for CaP and a two to eightfold increased risk for CaP-associated mortality6. More recently, a comprehensive review reported an association between BPH and CaP, suggesting that BPH is a risk factor for CaP7. While the possibility exists that BPH patients have an increased risk of developing CaP and CaP-associated mortality, it remains unclear how and why BPH patients are at risk for developing CaP.Benign prostatic hyperplasia (BPH) is a chronic urological disorder characterized by noncancerous enlargement of the prostate gland8. Epidemiological studies have reported Cd could be a potent prostate carcinogen because it is found in significantly higher levels in tissues and plasma of CaP patients than healthy controls9. Cd is an endocrine disruptor in experimental models10, supporting the hypothesis that this metal carcinogen can potentially induce the development of hormone-dependent tumors in humans, including that of the breast and uterus12. However, its molecular effects on malignant transformation of BPH cells remain elusive.Cadmium (Cd) is a known metal carcinogen and is one of the most abundant occupational and environmental pollutants found in air, soil, water, dietary products, and tobacco smoke13. ZIC2 plays a regulatory function in pluripotent and self-renewal of cancer stem cells (CSCs)15 and is also highly expressed and involved in tumorigenesis of various cancer types, including CaP16. As a zinc finger transcriptional factor, ZIC2 binds with the zinc finger domains of other protein families, including glioma-associated oncogene (GLI) in either a synergistic or antagonistic manner18. GLI proteins are also the downstream targets of the Sonic hedgehog (Shh) pathway that are an important therapeutic target for CaP treatment. Several studies have demonstrated growth arrest in CaP cell lines and xenografted tumors in mice20 following the inhibition of Shh signaling. Shh-associated secretory protein binds and inactivates patched1 (PTCH1), resulting in the release of smoothened protein and activation of GLI1, GLI2, and GLI3 receptors. GLI1 predominantly functions as a transcription activator, while GLI2 and GLI3 function as either an activator or repressor21. GLI1 activation initiates the expression of downstream target genes involved in proliferation (cyclin D1), survival , metastasis (Snail), and stem cell activation (Nanog and SOX2)22. Activated GLI1 and GLI2 proteins can also directly promote the expression of a group of genes involved in the process of epithelial mesenchymal transition (EMT)23.Deregulation of transcription factors, such as the zinc finger of cerebellum 2 (ZIC2) has been linked to heavy metal exposure and metal-induced carcinogenesisHere, for the first time, we report direct evidence that Cd exposure induces malignant transformation of BPH1 cells that exhibit an aggressive phenotype similar to that of CaP. In addition, our results suggest that chronic exposure of Cd to BPH1 cells was responsible for stem cell renewal, proliferation, and tumorigenesis of the transformed cells.2 at 37 °C in an incubator. The RWPE-1 cells were cultured in keratinocyte serum-free medium containing l-glutamine, Epidermal Growth Factor (EGF) and bovine pituitary extract (BPE).The BPH1 cell line was a kind gift from Dr. Simon W. Hayward . The cells were authenticated by Genetical Cell Line Testing . The cells were treated with 10 µM Cd  for 1 year and transformed into a malignant phenotype. The transformed cells were named Cd-transformed BPH1 (CTBPH1). Human normal prostate epithelial cells (RWPE-1) were purchased from the American Type Culture Collection . BPH1 and CTBPH1 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, and 1% antibiotic and antimycotic solution in a humidified atmosphere of 5% CO24. BPH1 cells following exposure to 10 µM Cd for 24, 48, and 72 h.Cell viability assays were performed using the trypan blue exclusion method or the 3--2, 5-diphenyltetrazolium-bromide (MTT) assay as described previously25. A group of at least 50 or more cells were considered as a colony and those colonies were counted.Colony formation assays were performed on Cd-exposed BPH1 and CTBPH1 cells to monitor anchorage-independent growth via the CytoSelectTM 96-well in vitro tumor sensitivity assay kit +, and ZIC2− using Boyden chambers equipped with polyethylene terephthalate membranes with 8-μm pores (BD Biosciences) as described previously26. Similarly, migration assays were performed for Cd-exposed BPH1 cells in six-well plates as described previously24.Invasion assays were performed on BPH1, 6-month Cd-exposed BPH1, CTBPH1 (12-month Cd-transformed cells), ZIC2+ and ZIC2− cells were generated by forced floating method using 96-well round-bottom Ultra Low Attachment plates . Single-cell suspension of ZIC2+ and ZIC2− cells at a density of 2 × 103 cells in 200 μl of respective culture media supplemented with MatrigelTM  was loaded into each well. Then, the morphology of the spheroids, cell growth were characterized over a 7-day culture period in triplicate.Spheroids of ZIC25 cells/well. After 24 h, the cells were transiently transfected with short-interfering RNA (siRNA) specific for ZIC2 or GLI1, or control siRNA, or an overexpression plasmid specific for ZIC2 for 48 h. Lipofectamine® 2000 was used as a transfection reagent. After 48 h, lysates were prepared and western blotting was performed.BPH1 cells were seeded in six-well plates at a density of 3 × 10xL (Santa curtz 7B2.5), X-linked inhibitor of apoptosis protein , cleaved caspase-3 , PTCH1 (ab53281), NOTCH1 , SOX2 , E-cadherin , N-cadherin , Slug , and β-catenin . Protein–antibody complexes were visualized using enhanced chemiluminescence as previously described25. For immunoprecipitation experiments, the protein samples were immunoprecipitated with ZIC2 antibody at 4 °C under agitation overnight following protein extraction using radioimmunoprecipitation assay (RIPA) buffer; the immunoprecipitated protein was then pulled down with protein A-agarose beads  at 4 °C under rotary agitation for 3 h. Immunoprecipitates were washed three times with RIPA buffer. After centrifugation, the pellets were resuspended in sample buffer and heated for 5 min at 95 °C for sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by immunoblot analysis.BPH1, transforming cells , and CTBPH1 cells were seeded in six-well plates and incubated for 24 h. Cell lysates were prepared, and western blotting was performed using specific antibodies against ZIC2 (ab150404), GLI1 (ab151796), Shh (ab53281), NANOG , ZIC2 immunohistochemistry (Sigma AU35821), CD44 , Poly(ADP-ribose) polymerase (PARP) , NFκB-p65 , Bcl2 , Bcl27.Total RNA was extracted from BPH1, Cd-exposed BPH1 , CTBPH1, and ZIC2 siRNA-treated CTBPH1 cells and RT-qPCR performed for ZIC2, SOX2, Nanog, CD44, and Notch1 expressions as described previously28. BPH tissue microarray and xenograft tumor tissues were examined for ZIC2, GLI1, and p65 expression using immunohistochemistry analysis.Immunofluorescence for ZIC2 and GLI1 expression was performed on Cd-exposed BPH1 and CTBPH1 cells, as described previously7) cells were incubated with 2.0 μl of fluorochrome-conjugated ZIC2 antibody before being sorted using fluorescence-activated cell sorting (FACS). Cells were analyzed on a Cytopeia Influx FACS using Spigot. Cell subpopulations were separated based on the surface antibody labeling and collected by discriminatory gating. After selecting for ZIC2+, cell suspensions were sorted from BPH cell population.Cd-exposed BPH1 or CTBPH1  in 50 μl Matrigel (Corning). The mice were monitored twice weekly, and the tumor volumes were measured once per week. At the end of experiments, the mice were euthanized via CO2 asphyxiation; the tumors were removed and fixed in 10% formalin for histopathological studies.Five-week-old male BALB/c (nu/nu) athymic mice were purchased from The Jackson Laboratory and housed in the University of Louisville vivarium under pathogen-free conditions. All experimental animals were maintained in accordance with the Institutional Animal Care and Use Committee approval and approval was obtained from the ethical committee of the University of Louisville, KY. The mice were randomly divided into four groups of eight each . At 7 weeks of age, the mice were subcutaneously injected with BPH1, CTBPH1, ZIC2t-test was performed for two-group comparisons and one-way analysis of variance analysis was performed for multiple group comparisons. The statistical significance was set at a P < 0.05, and values were presented as means ± SD.All statistical analysis used GraphPad Prism 8.0a software . An unpaired two-tailed Student’s XL, and XIAP but did not significantly alter the expression of proapoptotic proteins cleaved caspase-3 and PARP . Western blots and qRT-PCR confirmed that ZIC2 and Shh pathway markers were upregulated, validating our transcriptomic analysis. The results confirmed the activation of Shh signaling in Cd-exposed BPH1 cells, as determined by a decrease in PTCH1 expression and corresponding upregulation of Shh, GLI1, and ZIC2 Fig. . While CTo further confirm that this activation is specific to Cd-exposed BPH1 cells, we assessed ZIC2 levels in RWPE-1 cells following acute Cd exposure. No changes in ZIC2 mRNA and protein expression were noted in Cd-exposed RWPE-1 cells Fig. , suggestAlthough the use of immortalized cell lines is imperative for metal carcinogenesis especially for transformation assay, the effect of the immortalization process itself on the molecular signature of the metal carcinogen-induced transformation remains a concern for researchers. To address this, we compared effect on cell viability and ZIC2/GLI1 expression on primary human BPH cells with normal primary BPH cells following Cd exposure. The Cd exposure reduced the cell viability (not significantly) as seen in primary human BPH cells and increased the expression of ZIC2 and GLI1 Fig. . This coIn order to develop an in vitro model of Cd-induced malignant transformation, we chronically exposed BPH1 cells to 10 μM Cd for up to 16 months. We periodically performed anchorage-independent growth assays to determine the malignant transformation of Cd-exposed cells. BPH1 cells began forming colonies after 6 months of Cd exposure, and the number of the colonies increased with exposure time. This confirmed that Cd exposure induced the malignant transformation of BPH1 cells Fig. .Fig. 3EfWe next performed western blot analysis to confirm whether the molecular signature of chronic Cd exposure was similar to acute exposure in the BPH1 cells. The results showed that similar to acute exposure, chronic Cd exposure also increased the expression of ZIC2, Shh, and GLI1. Upregulation of prosurvival proteins p65, XIAP, and Bcl2 stem cell markers Nanog and CD44 was also observed Fig. . InteresAlthough, the BPH1 cells were chronically exposed to Cd up to 16 months to induce malignant transformation, we did not observe any significant phenotypic changes in the cells exposed beyond 12 months. Thus, we considered the cells exposed to Cd for 12 months to be completely transformed and will refer to them as CTBPH1 below.30. We used FACS to sort the ZIC2-negative and -positive cell populations at specific time points  to determine whether Cd-mediated ZIC2 CSCs properties in the transformed BPH1 cells. A gradual increase in the percentage of ZIC2+ cells was observed in the transforming cells; the 12-month timepoint had the highest fraction of positive cells (45–50%; Fig. + cells formed spheres while ZIC2− cells did not form spheroids (Fig. + developed tumors versus mice injected with ZIC2− cells (Fig. + and ZIC− CTBPH1 cells. Increased, MMP 9, slug and β-catenin expression followed by downregulation of E‐cadherin was noted in the ZIC+ cells as compared with ZIC− CTBPH1 cells. (Fig. Self-renewal, higher tumorigenicity, sphere formation, and metastatic ability are characteristic properties of CSCsids Fig. , confirmlls Fig. . To furtlls Fig.  ZIC2 ovels. Fig. .Fig. 4ZI+ cells showed increased invasive and migratory properties (Fig. − cells. These results suggest that ZIC2 may aid in the transformation of Cd-exposed BPH1 by inducing mesenchymal phenotype and promoting stem cell renewal.Furthermore, invasion and migration assays demonstrated that ZIC2ies Fig.  versus ZConsidering that Cd-mediated ZIC2 activation promoted the self-renewal properties of the CTBPH1 cells, we inhibited the ZIC2 expression in these cells to determine if this altered their self-renewal potency. Spheroid assays showed that silencing ZIC2 expression in CTBPH1 cells completely abrogated the formation of spheroids versus cells transfected with scrambled vectors Fig. . SubsequAs shown above, Cd exposure induced ZIC2- and Shh-mediated GLI1 signaling in the CTBPH1 cells. We postulated that the induction of prosurvival or inhibition of proapoptotic signaling may be regulated by activation of GLI1. Interestingly, cell viability assays showed that inhibition of GLI1 inhibited the growth of CTBPH1 cells Fig. . To conf31; thus, we performed IPs to confirm whether Cd-induced BPH carcinogenesis occurs due to the interaction between these two proteins. Unlike the vehicle-treated BPH1 cells that showed a complete absence of GLI1/ZIC2 binding, strong binding was observed in CTBPH1 cells (Fig. These results indicate that both ZIC2 and Shh/GLI1 signaling may be necessary for Cd-induced transformation of BPH1 cells. To delineate how the GLI1 and ZIC2 molecularly interact and coordinate their functions during Cd-mediated transformation, we first investigated the localization of these two transcription factors in the CTBPH1 cells. Western blot and immunofluorescence analysis revealed that Cd exposure caused induction and nuclear accumulation of ZIC2 and GLI1 versus vehicle-treated control cells Fig. . Previoulls Fig. . These rNext, we investigated whether CTBPH1 cells could induce tumor formation in nude mice. We observed a progressive increase in tumor growth in mice injected with CTBPH1 cells versus mice injected with vehicle-control cells Fig.  over a 4The immunohistochemistry of BPH tissue microarrays comprising tissue sections from 80 BPH patients showed that ZIC2 immunostaining was predominant in BPH specimens versus normal prostate tissue Fig. . Examina32, the underlying mechanisms involved in Cd carcinogenesis remain unclear. Here, we demonstrated that long-term chronic exposure to Cd to BPH1 cells induced transcriptional changes responsible for stem cell renewal, proliferation, and tumorigenesis of the transformed CTBPH1 cells. Cd-mediated ZIC2 activation-initiated stem cell renewal and activation of GLI1: both of these steps promoted cell proliferation of the transformed cells. Thus, we confirmed that the dynamic interaction of ZIC2 and GLI1 are necessary for Cd-induced malignant transformation of BPH cells, a phenomenon not observed in normal prostate epithelial cells.Although Cd is a well-known human prostatic carcinogen34. Similarly, this study helped show that chronic exposure to Cd resulted in malignant transformation of BPH1 cells. We previously showed that defective autophagy was responsible for Cd-induced transformation of RWPE-135. Here, ZIC2 and GLI1 is responsible for Cd-inducted transformation in BPH1 cells, and it appears that it is specific to BPH1 cells and not to normal prostate epithelial cells.We previously demonstrated the implantation of Cd-transformed RWPE-1 cells; these cells formed tumors in xenograft models36. Similarly, the stem cell renewal function of ZIC2 was established37 by activating the stem cell transcription factors, such as Oct4, Sox2, and Nanog as binding sites38. We also found that Nanog and CD44 were upregulated in CTBPH1 cells as compared with vehicle-treated cells. Previous studies demonstrated that CSCs may be responsible for tumor initiation, invasion, distant metastasis, chemo-resistance, self-renewal, and differentiation potential39. Thus, we set out to isolate ZIC2+ cells from CTBPH1 cells and characterize the ZIC2 function. CSCs are believed to be able to form spheres in culture that can then exhibit extensive similarities to endogenous CSCs in human tumor tissues40. Here, sphere formation assays using ZIC2+ and ZIC2− cells revealed the increased ability of ZIC2+ cell to form spheroids, suggesting that ZIC2 activation imparts CSC properties to Cd-exposed BPH1 cells. Interestingly, upon testing the in vivo potency of ZIC2+ versus ZIC2− cells in athymic mice, we observed tumor formation in mice injected with ZIC2+ cells but not in those injected with ZIC2− cells. However, our results also demonstrated that the inhibition of ZIC2 expression did alter the expression of cell survival and proapoptotic markers, suggesting activation of an alternative machinery in Cd-exposed cells.Earlier studies established the oncogenic role of ZIC2 in pancreatic ductal adenocarcinoma cells by activating the expressions of FGFR3 and ANXA814 demonstrated that the overexpression of ZIC2-induced nuclear retention of GLI1: the downstream effector of oncogenic Shh signaling. Other studies have suggested that GLI1 physically interacts with ZIC2 protein via a zinc finger domain42. We found that Cd-induced Shh ligands that in turn inhibited PTCH1 expression leading to the activation of GLI1 and downstream prosurvival targets, such as Bcl2, BclXL, and XIAP. This strongly suggested that activation of Shh signaling initiated the prosurvival machinery and promotion of cell proliferation in Cd-exposed BPH1 cells; this was later corroborated when we found that silencing GLI1 expression inhibited cell proliferation in CTBPH1 cells. Interestingly, we also noted that the interaction of ZIC2 and GLI1 was stronger in CTBPH1 cells than RWPE-1 and BPH1 cells. This suggests that the oncogenic effects of ZIC2 could be due to the interaction between these two proteins and could in turn aid in the tumorgenicity of the CTBPH1 cells.Recently Chan et al.44. Wei and Shaikh45 reported that prolonged Cd treatment in triple-negative breast cancer cells stimulates cell proliferation, adhesion, cytoskeleton reorganization, as well as migration and invasion. Here, we observed that both ZIC+ cells and chronic exposure to Cd-induced expression of mesenchymal marker, β-catenin. The Wnt/β-catenin pathway plays a pivotal role in multiple malignancies, including regulating cell proliferation, EMT, and migratory process47. Moreover, Wnt/β-catenin signaling could also regulate Zic gene expression48. Thus, our results strongly suggest that chronic Cd exposure promotes invasion, migration, and EMT in BPH1 cells via activation of the Wnt/β-catenin signaling pathway.Recent studies have demonstrated that Shh activation plays a central role in EMT regulation, involving the loss of cell–cell adhesion, changes in cell morphology, and the propensity to migrate and invade CaPIn summary, we found that both exposure of Cd in BPH1 cells causes malignant transformation and tumorigenesis via upregulation of Shh/GLI signaling, as well as ZIC2 activation. We also found that both ZIC2 and GLI1 function as complementary signals initiating stem cell renewal and leads invasion as well as migration, respectively. Thus, they are necessary components for Cd-induced prostate carcinogenesis.


Primary neurons from rodent brain hippocampus and cortex have served as important tools in biomedical research over the years. However, protocols for the preparation of primary neurons vary, which often lead to conflicting results. This report provides a robust and reliable protocol for the production of primary neuronal cultures from the cortex and hippocampus with minimal contribution of non-neuronal cells. The neurons were grown in serum-free media and maintained for several weeks without any additional feeder cells. The neuronal cultures maintained according to this protocol differentiate and by 3 weeks develop extensive axonal and dendritic branching. The cultures produced by this method show excellent reproducibility and can be used for histological, molecular and biochemical methods. A primary neuron culture from embryonic rodent hippocampus or cortex has been one of the most fundamental methodologies for modern neurobiology. Primary neurons can be easily cultured and over a few days or weeks differentiated into neurons with clearly separable axons, dendrites, dendritic spines and synapses. By modifying the culture medium and conditions, numerous factors responsible for directing different aspects of neuronal survival, differentiation and phenotype have been revealed . Earlier One of the disadvantages of primary culture is that they do not divide in culture and need to be generated from embryonic or early postnatal brains every time. Moreover, successful dissection and preparation of cultures require substantial skill and experience. Over several decades, cell lines have been discovered and created that mimic many or most of the features of primary neurons ,12. MoreNeuronal cultures, however, vary vastly depending on source, age of derivation and culture conditions. Results obtained by a culture protocol used in one lab may not be reproducible in another lab, which adds to the ongoing discussion about reproducibility crisis. We have over more than a decade developed and refined a culture protocol of primary neurons derived from E17–18 rat embryos, which has been successfully used in several publications ,16. Heread libitum. All the experiments were performed at the Animal Unit, Biomedicum, University of Helsinki. The procedures were followed according to institutional guidelines by the University of Helsinki internal license number: KEK17-016.Pregnant female Wistar rats were obtained from Envigo . The plug date of the female rats was marked E0. All embryos staged at E17–18 from the female rats were used in the experiments. The embryos were staged according to the Witschi Standard Rat Stages. The average litter size was 9 pups per female rat. Animals were kept in standard conditions . Food and water were available 80 g NaCl,0.2 g KCl,2HPO4 × 2H2O,14 g Na2PO4.2 g KH2O and autoclaved at 121°C for 20 min.Make upto 1 l with Milli-Q HPhosphate-buffered saline (PBS) buffer pH = 7.4HBBS ,1 mM sodium pyruvate,10 mM HEPES.Preparation medium, pH = 7.2DMEM,10% fetal bovine serum,l-glutamine,1% 1% penicillin–streptomycin.Dulbecco’s modified Eagle’s medium (DMEM) ++1 mg DL-Cysteine HCl,1 mg bovine serum albumin (BSA),25 mg Glucose in 5 ml PBS.The stock Papain buffer stored at −20°C0.5 mg papain,10 μg DNase I in 5 ml Papain Buffer.Papain solution10 μg DNase I in 10 ml Preparation medium as above.Trituration mediumNeurobasal medium,2% B27 supplement, L-glutamine,1%1% penicillin–streptomycin.Growing mediuml-lysine working solution was made as 1:10 dilution of stock Poly-l-lysine in Milli-Q H2O.Poly-40 g PFA in 1 l PBS [The final solution was filtered with a Whatman filter paper (pore size 45 mm).4% Paraformaldehyde (PFA)0.3% Triton X-100 in PBS.PBST1% BSA,4% normal goat serum,0.3% Triton X-100 in PBS.Blocking bufferPrimary antibodies used were glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN) and microtubule-associated protein 2 (Map2)  at 1:100Secondary antibodies were goat-anti-rabbit 647, goat-anti-mouse 568 and goat-anti-chicken 488 diluted 1:1000 in blocking buffer.2) in a CO2 euthanasia chamber. Abdominal skin was washed with 70% ethanol followed by an incision to cut and open the peritoneal cavity (Supplementary video S1). Amniotic sacs were exposed with fine scissors and embryos were taken out from the uterus. These embryos were transferred to a 50-ml polypropylene Falcon tube with 30 ml of PBS on ice. The embryos were transferred to a sterile laminar hood where further processing took place. The heads were decapitated by scissors and immediately transferred to ice-cold PBS stored on ice. Brain dissection was performed in 10-ml preparation medium [The pregnant female Wistar rats were terminally anesthetized with carbon dioxide CO in a CO2The dissected hippocampi were transferred into 10 ml of preparation medium  on a 35-The hippocampi were transferred using a fire-polished glass Pasteur pipette into a 15-ml polypropylene Falcon tube with 5 ml of papain buffer  with addThe tissue was incubated for 10 min at 37°C. After the tissue had sunk to the bottom, excess papain solution was discarded using a glass pipette.Pipette 3 ml of trituration medium  to the tThe cells were triturated in the trituration medium  with a fThe trituration was repeated for three times with a fire-polished glass Pasteur pipette.g in Centrifuge 5810 (Eppendorf) at RT.The pooled supernatant with the dissociated tissue from each trituration step (approximately 9 ml) was centrifuged for 5 min at 900 rpm/154×The clear supernatant was discarded.The remaining cell pellet was re-suspended in 1 ml of fresh growing medium at RT .The hippocampal cells were diluted to 1:10 (5 μl of cells + 45 μl growing media). This gentle dissociation method results in hippocampal cells with very few dead cells or debris. Therefore, diluted cells were directly used for cell counting. Ten microliters of the diluted cells were transferred into a disposable hemocytometer by a pipette. For cell counting, cells in four fields of the hemocytometer were counted using a manual cell counter under a Leica stereo microscope and averaged.Hippocampi from ten embryos typically yields 5–6 million neurons.2. The cells were grown at 37°C, 5% CO2 humidified incubator  for the indicated times  on to each plate.2 humidified incubator for 30 min. During this incubation, glial cells and other unwanted debris adhere to the bottom of the plate and the cortical cells are also recovering from the trituration.The plates were incubated at 37°C, 5% COPipette 7.5 ml of DMEM++ on to uncoated 90 mm Petri dishes. Additionally, pipette 2.5 ml of the supernatant from the previous step (representing five brains) on to each new Petri dish or dishes.g in RT.The supernatant was carefully removed, without disturbing attached glial cells and unwanted cell debris at the bottom of the plate into a new 15-ml Falcon tube using a pipette. The collected supernatant from the above step was centrifuged for 5 min at 900 rpm/154×After centrifugation, the supernatant was discarded and the pellet was re-suspended in 1.5 ml growing medium  per eachThe cell suspension was removed into a new 15-ml Falcon tube using a pipette.The cortical cells obtained by this method results in a lot of cell debris. Therefore, cell counting for the cortical cells was performed along with 0.4% of Trypan Blue dye. The cortical cells were used at 1:20 dilution (5 μl of cell suspension + 75 μl growing medium + 20 μl Trypan Blue) for cell counting. The cell counting was done similarly as for the hippocampal cells.Cortices from ten embryos typically yield 50–60 million neurons. If the number of embryos is low, the relative yield is typically lower than when the cells are prepared from a large number of embryos.l-lysine [l-Lysine. After at least 18 h (overnight) of incubation and before plating the cells, the plates were rinsed twice with 500 µl of PBS.The multi-well plates  or round coverslips (histochemistry) within a 4- or 24-well plate were pre-coated for 18 h with 500 µl of 10 μg/ml Poly-2 in a humidified incubator . Half of the growing medium was changed once a week.The number of cells plated per well varies depending on the downstream processing of the samples. Representative examples of plating densities for different cell types and well sizes are documented in 2 on the coverslip in a four-well plate. The cells were grown at 37°C, 5% CO2 in a humidified incubator. The cell plating density always depended on the purpose of the experiment, examples of typical cell plating densities are shown in the The four-well plates were washed three times for 5 min with 200 µl of PBST .The fixed cells were pre-blocked with 200 µl blocking buffer  for 1 h After blocking, 150 µl of primary antibody  diluted After incubation with the primary antibody, the buffer was removed and cells were washed 3 × 10 min with 200 µl of PBST.The secondary antibody  was diluAfter the incubation with the secondary antibody, the coverslips were washed for 10 min in 200 µl of PBST followed by two times for 10 min with 200 µl of PBS.2O to rinse off the PBS. Before mounting, coverslips were gently dried by touching the edge on to a tissue paper.The coverslips were held with fine forceps and dipped into 50 ml Falcon tubes containing Milli-Q HThe cells attached on to the coverslips were mounted on the Superfrost slides with 10 µl of mounting media . Two covThe slides were stored in dark, protected from light at 4°C until imaging.For immunostaining, we used 26000 cells/cmhttps://imagej.nih.gov). Cells from a single focal plane were analyzed for an area of 4 mm2 per image per coverslip. We analyzed five images per coverslip and four coverslips for each time point was used for imaging. Thus, 20 images for each time point was analyzed.The whole slide imaging was performed using a 20× objective in a Histoscanner  at the genome biology unit, Biomedicum Helsinki. The images were analyzed using panoramic viewer software . Four coverslips for each stage were scanned. Cell counting was performed using ImageJ software . The groups were compared using one-way ANOVA. All the data are represented as means ± SEM.The method for culturing primary neurons described here has been developed and used in our lab for over two decades. The quality of neurons has been high and reproducible over the period of time. The overall procedure is outlined in 2 for both hippocampal and cortical cells. In the cortical cell preparations, no GFAP positive cells were detected at 7 and 14 days in vitro (DIV). At 21 DIV the GFAP cells formed 4% of the total cell population in the primary cortical cells , GFAP  and Map2  at three different time points . The denal cells A. The nual cells B. The deal cells .The protocol described here is originally based on the protocol described by Brewer and Cotman , with sol-Lysine. For histochemical staining and live imaging, the cells were grown on glass coverslips.The primary neuronal cell culture is a standard system for the investigation of neuronal structure and function at a high resolution. The current protocol generates relatively pure neuronal cultures with maximum reproducibility and minimal contribution of glial cells. In this method we have cultured the neurons for 3 weeks without any additional feeder cells. For cell dissociation, we have used papain only for hippocampal cells rather than trypsin, while cortical cells were dissociated by trituration without any prior enzymatic digestion. It has been observed that trypsin digestion of tissue leads to RNA degradation . The corEspecially for biochemical assays, such as transcriptomic and proteomic analyses, it is important to control and minimize the number of other cell types in the culture, particularly the number of glial cells. In cortical cultures, we found virtually no glial cells at all during the first 2 weeks in culture and less than 5% at 21 DIV, and also in hippocampal cultures, the number of glial cells was low until DIV 14, but started to increase thereafter. This is comparable with reports using related protocols that have reported less than 5%  or virtuin vitro models for biomedical research of the post-mitotic neuronal cells. The primary cells are genetically more stable than neuronal cell lines and they maintain in culture many crucial markers and functions as seen in vivo. Thus, they complement in vivo experiments allowing for more controlled manipulation of cellular functions and processes. Once neurons are cultured, advanced molecular and biochemical studies can be easily performed. For example, successful CRISPR-cas9 gene editing has been achieved using primary neuronal cultures [The cell lines have been the largest source for medical research due to their immortal nature. These immortal cell lines have produced variable results arising, among others, from different passage times leading to genetic drift and selection for the faster growing cells . The pricultures . FurtherThe preparation of cells should be performed within no more than 2–3 h after dissecting the pups.Tissues should be preserved on ice all the time to prevent degradation.Petri dishes used are a common source of problems in the primary cell culture. We have reported here the manufacturers and plate types that have worked in our hands. Our previous experience with other manufacturers has resulted in low yield and bad quality of neurons with excessive glial contamination.Trituration of cortical and hippocampal tissues should be gentle without any force. Higher force results in a large number of dead cells detected in the hemocytometer counting. If the neuron quality is poor or yield is half of the average, we obtain typically using this method, these neurons should not be used for any downstream processing.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Results: Ad interim knockdown of HIF-1α also inhibited manganese-dependent superoxide dismutase (MnSOD), catalase and sestrin 3 (Sesn3) expression in OS cells. Furthermore, hypoxia-induced ROS formation and apoptosis in OS cells were associated with CYP450 protein interference and were ablated by HIF-1α silencing via siRNA. Conclusions: Our data reveal that HIF-1α inhibits ROS accumulation by directly regulating FoxO1 in OS cells, which induces MnSOD, catalase and Sesn3 interference, thus resulting in anti-oxidation effects. The combination of an HIF-1α inhibitor  and ROS inducer  can prohibit proliferation and migration and promote apoptosis in MG63 cells in vitro while inhibiting tumour growth in vivo.Background: The present study was designed to explore the underlying role of hypoxia-inducible factor 1α (HIF-1α) in reactive oxygen species (ROS) formation and apoptosis in osteosarcoma (OS) cells induced by hypoxia. Methods: In OS cells, ROS accumulated and apoptosis increased within 24 h after exposure to low HIF-1α expression levels. A co-expression analysis showed that HIF was positively correlated with Forkhead box class O1 (FoxO1) expression and negatively correlated with CYP-related genes from the National Center for Biotechnology Information’s Gene Expression Omnibus (NCBI GEO) datasets. Hypoxia also considerably increased HIF-1α and FoxO1 expression. Moreover, the promoter region of FoxO1 was directly regulated by HIF-1α. We inhibited HIF-1α via siRNA and found that the ROS accumulation and apoptosis induced by hypoxia in OS cells decreased. In this study, a murine xenograft model of BALB-c nude mice was adopted to test tumour growth and measure the efficacy of 2-ME + As Osteosarcoma (OS) is the most common primary bone cancer and is one of the leading causes of mortalities related to cancer in paediatric patients . The fivThe subgroup of Forkhead box class O (FoxO) transcription factors contains important regulators of the genome; they are characterized by the structural feature of a winged-helix in their DNA binding domain ,5. The mMitochondria are centres for cellular bioenergetic activities and are important sources for ROS . ROS proSaccharomyces cerevisiae and mitochondrial transcription specificity factors, in up- and downstream signalling pathways. All of the above transcription factors bind to and are further regulated by nuclear respiratory factors and PGC-1 family coactivators [The genome within the mitochondria encodes 12 proteins closely associated with biogenetic activities . The exptivators ,21. In ativators . InteresWe have previously reported that FoxO1 could promote the expression of antioxidant proteins such as MnSOD, catalase and Sesn3 . Specifi2O3 can inhibit MG63 cell proliferation and migration while promoting MG63 cell apoptosis and intracellular ROS accumulation. To further examine the effect of 2ME + As2O3, a xenograft murine model of OS in BALB/c nude mice was used to test its efficacy. In an in vivo drug-sensitivity test, the combination of 2ME and As2O3 achieved anti-tumour effects without obvious adverse reactions.Here, we present a comprehensive analysis of the transcriptional response to HIF-1α, revealing the repression of numerous nuclear-encoded mitochondrial genes through the regulation of FoxO1 function. We demonstrate that through this signalling arm, HIF-1α reduces cellular ROS production, independent of MnSOD, catalase and Sesn3 activation. Regulation of mitochondrial structure and function could be an important role for HIF-1α factors in regulating ROS production, and these processes can affect cellular adaptation to hypoxia. Through in vitro drug experiments, we found that 2ME combined with AsWe retrieved microarray data for normal tissues  and human osteosarcoma tissues  from the National Center for Biotechnology Information’s Gene Expression Omnibus (NCBI GEO) datasets for a total of eight samples.Ethical approval: This study was approved by the Ethics Committee of Fudan University Shanghai Cancer Center.In all, 29 paired osteosarcoma specimens and adjacent normal bone tissues, which were confirmed as primary malignant bone cancer by trained pathologists, were collected from the Department of Musculoskeletal Oncology of the Fudan University Cancer Hospital  in 2017–2018. One of these samples was immediately snap-frozen in liquid nitrogen. The other tissues were formalin-fixed and paraffin-embedded.Paraffin-embedded blocks were cut into 4 μm thick sections and dewaxed and hydrated. Then, the slices were immersed in distilled water containing 3% hydrogen peroxidase twice to reduce endogenous oxidase activity. Afterwards, the tissue sections were incubated with primary antibodies for 2 h at room temperature, and a secondary antibody was subsequently applied to the cells at room temperature for 40 min. The staining degree was determined by diaminobenzidine (DAB) chromogen . Subsequently, the tissues were dehydrated and sealed with gum. Five random fields of view (100×) were captured with a camera and a microscope .2 (5%) atmosphere.Two human OS cell lines (U2OS and MG63) were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum , 100 U/mL penicillin and 100 mg/mL streptomycin (Thermo Fisher Scientific). Regular osteoblast cells (hFOB1.19), used as a control, were acquired from the Chinese Cell Bank of the Chinese Academy of Sciences  and cultured in Ham’s F12/DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. The cultures were preserved at 37 °C in a humidified CO® RT Master Mix Perfect Real-Time Kit  and SYBR Green Master Mix . qPCR was performed on an Applied Biosystems 7900HT Real-Time System .Complete RNA was extracted from cells and tissues with Trizol reagent  according to the protocol. All mRNA was subjected to quantitative polymerase reaction and reverse transcription according to the protocols for the PrimeScriptCollected cells were lysed with RIPA protein extraction reagent  containing a protease inhibitor cocktail . The lysates were then loaded onto sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gels for separation, transferred to polyvinylidene fluoride (PVDF) membranes and blocked in 5% milk prior to incubation with the indicated primary and secondary antibodies. Autoradiograms were quantified through densitometry, and GAPDH was used as a control. The antibodies against HIF-1α, FoxO1, MnSOD, catalase and Sesn3 were purchased from Cell Signaling Technology .® HD transfection reagent  according to the manufacturers’ instructions, respectively. The cells were then collected to observe the knockout or overexpression efficiency via qRT-PCR for 48 h after transfection. Two distinct siRNAs against FoxO1 were created and synthesized by GenePharma . The objective si-FoxO1 sequences and synthetic FoxO1 sequence (3099 bp) are described in previous research (15). The siRNA sequence targeting FoxO1 (si-FoxO1) was 5′-GCTCAACGAGTGCTTCATCAAGCTACCCA-3′.U2OS and MG63 cell lines were transiently transfected with siRNAs after being cultured in six-well plates overnight. A mixed negative control, a plasmid overexpressing FoxO1 and a blank vector were used as well with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) and FuGENE3 cells were seeded in quadruplicate for each group in a 96-well plate. The cells were incubated with 10% CCK-8 reagent  diluted in regular culture medium at 37 °C until optical colour conversion occurred. Proliferation rates were measured at 24, 48 and 72 h after transfection. The absorbance of each well was determined with a microplate reader  set at 450 nm.Cell viability was determined with a CCK-8 assay. First, 1 × 10Four- to six-year-old female nude mice were purchased from Vital River Laboratory Animal Technology . All animals were housed in individual ventilated cages, provided sterilized water and food at libitum and handled under specific pathogen-free conditions in the Institute’s animal care facilities, which meet international standards. The mice were checked for their health status, animal welfare supervision was provided and experimental protocols and procedures were reviewed by a certified veterinarian. All animal experiments were carried out in accordance with the Chinese governing law on the use of medical laboratory animal .2O3/DMSO solution and a 2-ME + As2O3/DMSO solution (2-ME at 5 mg/kg and As2O3 at 5 mg/kg). 2-ME was administered once every two weeks, and As2O3 was administered for five subsequent days and then every two days. The length of the treatment course was three weeks. Food and water were supplied ad libitum after treatment. The health status was monitored by a specific veterinarian. All animals were euthanized and sacrificed by cervical dislocation after treatment. After two courses of treatment, the mice were euthanized, and the tumours were removed and weighed.In total, 32 female mice were divided randomly into four groups. The average weight of the animals was 12 grams. The administered drugs were dissolved in dimethyl sulfoxide (DMSO). For the control group, the animals were injected with only DMSO intraabdominally. The other three groups were injected with a 2-ME/DMSO solution, an Ast-test or one-way analysis of variance (ANOVA). Recurrence-free survival and total survival were determined by Kaplan–Meier survival analysis and compared via log-rank test. For this study, p-values <0.05 were considered statistically significant. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed via the Database for Annotation, Visualization and Integrated Discovery (DAVID) program.All statistical analyses were executed with SPSS 22.0 software  and GraphPad Prism 5.0 . Differences between groups were analysed utilizing Student’s ΔΔCt values of HIF-1α and FoxO1 were significantly increased in bone cancer tissues relative to normal adjacent tissues (p < 0.05)  and osteosarcoma tissues  from NCBI GEO datasets through R Package Limma and Affy. In all, 17 genes were discovered to be significantly differentially expressed in these datasets A. Among  < 0.05) A. This f < 0.05) B. Intere < 0.05) .Pyruvate dehydrogenase (PDH) activity is inhibited through HIF-1α by upregulation of the target molecule, pyruvate dehydrogenase kinase (PDK-1). HIF-1α blocks pyruvate in the tricarboxylic acid cycle, thereby inhibiting mitochondrial oxidative phosphorylation. Because mitochondrial respiration is the main source of ROS, we hypothesized that HIF-1α could reduce ROS production. Intracellular ROS levels were significantly lower in MG63 cells treated with HIF-1α siRNA than in control cells A,B. In aHomo sapiens FoxO1 5′-UTR are shown in To further explore the role of HIF-1α in FoxO1-induced ROS changes, the effects of HIF-1α on FoxO1 expression were examined. Two hypoxia-responsive elements within the promoter region of FoxO1 were identified, indicating that FoxO1 was a potential HIF-1α target. The target sites in the The antioxidant proteins MnSOD, catalase and Sesn3 have been reported as primary downstream messengers of Akt/FoxO1 signalling . To elucCyp2c38, Cyp2c38, Cyp2c38, Ptgs2 and Alox12) participating in ROS metabolism were significantly increased in HIF-1α-silenced OS cells  and proliferation (CCK8) assays were performed on MG63 cells. A Transwell assay revealed that the migratory capabilities of MG63 cells were greatly decreased when they were treated with 2ME + As2O3 , which then leads to OS cell apoptosis. This double consequence of FoxO1 appears to be crucial in hypoxic injury regulation in OS cells. The combination of As


In their meta-analysis of observational studies, Low et al. showed a high prevalence of burnout syndrome (BOS) among medical and surgical residents across the globe with an aggregate prevalence of burnout as 51.0% (CI: 45.0–57%). However, the sample size in many of the included studies was quite low (only 26 out of 47 included studies had a sample size of more than 100 participants), and almost all of the 47 studies reported a rate of respondents of less than 80% . Furthermore, in many of them, the rate of respondents was unknown (5 out of 47) or less than 50% of eligible persons (23 out of 47 studies). As BOS is a self-reported syndrome, healthcare professionals who decided to participate in those studies were many of those affected by BOS, making the percentage of respondents potentially overstated due to the nonresponse bias. Policy decision-making in public health relies on evidence-based research; therefore, quality evaluation of studies in meta-analysis is essential to draw useful data for policymakers. Policy decision-making in public health relies on evidence-based research. Burnout syndrome (BOS) is a worrying phenomenon among clinicians and residents, not only in the United States, but across the entire globe . HoweverTherefore, the meta-analysis  of obserIn every systematic review and meta-analysis, however, the quality evaluation of included studies is essential to drawing useful data to be translated by policymakers into public health decisions. We have noted in this meta-analysis that Low et al. adopted the National Institute of Health’s Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies (NIH-QAT) , which iAs BOS is a self-reported syndrome, participants who responded were probably also those affected by BOS, potentially making the percentage of respondents higher due to the nonresponse bias; that is, the error resulting from distinct differences between the people who responded to a survey versus the people who did not respond . The pre